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Apatite nanocomposites with graphene (G) or graphene oxide (GO) nanoflakes, as well as with related carbonaceous materials, present promising applications in hard tissue engineering, biomedicine, or drug delivery. Different methodologies have been explored in the last years to prepare apatite-based nanocomposites. Sitting drop vapour diffusion (SDVD) methodology induces the heterogeneous nucleation of biomimetic apatite on the reinforcement material, improving biological properties of the nanocomposites. In this work SDVD was used to prepare G-apatite and GO-apatite nanocomposites. Prior to the SDVD experiments, G flakes were obtained by sonication-assisted liquid-phase exfoliation (LPE) using L-Alanine (L-Aln) as dispersing biomolecule, while a commercial aqueous Graphene Oxide (GO) dispersion was used for the nucleation essays in presence of the same biomolecules. A parallel set of nucleation experiments was performed in presence of Tb3+ ions, to endow the nanocomposites of luminescent properties. Characterization by XRD, FTIR, and TEM demonstrated the heterogeneous nucleation of needle-shaped apatite nanocrystals on the surfaces of G and GO flakes. Fluorescence spectroscopy certified the presence of Tb3+ ions in the nanocomposites resulting in luminescent materials which can be used in imaging or theragnostic. Finally, in vitro tests with human mesenchymal stem cells revealed excellent cytocompatibility and cell proliferation in presence of the nanocomposites.

Self-assembly and mineralization of type I collagen (Col) with nanocrystalline apatite (nAp), by adding a solution of Ca(OH)2 to a stirred Col-H3PO4 solution by fast dripping, allowed the preparation of Col/nAp fibrils with good crystallographic control of the mineral phase. In this work, in addition, we have cross-linked the mineralized fibers by using different reagents, namely glutaraldehyde (GTA), tannic acid (TA), 1-Ethyl-3-(3-dimethyl aminopropyl)-carbodiimide combined with N-Hydroxysuccinimide (EDC/NHS), and genipin (GP), aimed at producing different types of biopolymeric Col/nAp-based drug delivery scaffolds. In parallel, we have investigated two different methods to impregnate the scaffolds with molecules of the cocrystal diclofenac-metformin (DF-MET). The result, when using TA as a crosslinking reagent, shows the sequence of mineralized fibrils impregnation followed by crosslinking leads to maximum cocrystal molecule loading. The impregnated material is expected to be useful in settings with excessive and prolonged inflammation, since they affect negatively the fracture healing/bone repair processes, especially during the early stages of healing.

Maslinic acid (MA) is a plant-derived, low water-soluble compound with antitumor activity. We have formulated MA in the form of solid lipid nanoparticles (SLNs) with three different shell compositions: Poloxamer 407 (PMA), dicarboxylic acid-Poloxamer 407 (PCMA), and HA-coated PCMA (PCMA-HA). These SLNs improved the solubility of MA up to 7.5mg/mL, are stable in a wide range of pH, and increase the bioaccessibility of MA after in vitro gastrointestinal (GI) digestion. Gastrointestinal digested SLNs afforded MA delivery across in vitro gut barrier models (21 days old Caco-2 and mucus-producing Caco-2/HT29-MTX co-cultures). The cellular fraction of Caco-2/HT29-MTX co-cultures retained more MA from GI digested PCMA-HA than the Caco-2 monolayers. The concentration of MA reached in the basolateral chamber inhibited growth of pancreatic cancer cells, BxPC3. Finally, confocal microscopy images provided evidence that Nile Red incorporated in MA SLNs was capable of crossing Caco-2 monolayers to be taken up by basolaterally located BxPC3 cells. We have demonstrated that SLNs can be used as nanocarriers of hydrophobic antitumor compounds and that these SLNs are suitable for oral consumption and delivery of the bioactive across the gut barrier., Ministerio de Ciencia e Innovacion (MCIN/AEI/FEDER) RTI2018-101309-B-C21 RTI2018-101309-B-C22, European Molecular Biology Organization (EMBO-short term fellowships) 8475, Enterprise Ireland (Career-FIT), European Commission 713654, MF2018-0151 Food-BIBS, Science Foundation Ireland, Department of Agriculture, Food and Marine on behalf of the Government of Ireland 16/RC/3835, European Commission European Commission Joint Research Centre European Regional Development Funds of the Junta de Andalucia, Spanish Government

The complexity of the tumor microenvironment (TME) together with the development of the metastatic process are the main reasons for the failure of conventional anticancer treatment. In recent years, there is an increasing need to advance toward advanced in vitro models of cancer mimicking TME and simulating metastasis to understand the associated mechanisms that are still unknown, and to be able to develop personalized therapy. In this review, the commonly used alternatives and latest advances in biofabrication of tumor-on-chips, which allow the generation of the most sophisticated and optimized models for recapitulating the tumor process, are presented. In addition, the advances that have allowed these new models in the area of metastasis, cancer stem cells, and angiogenesis are summarized, as well as the recent integration of multiorgan-on-a-chip systems to recapitulate natural metastasis and pharmacological screening against it. We also analyze, for the first time in the literature, the normative and regulatory framework in which these models could potentially be found, as well as the requirements and processes that must be fulfilled to be commercially implemented as in vitro study model. Moreover, we are focused on the possible regulatory pathways for their clinical application in precision medicine and decision making through the generation of personalized models with patient samples. In conclusion, this review highlights the synergistic combination of three-dimensional bioprinting systems with the novel tumor/metastasis/multiorgan-on-a-chip systems to generate models for both basic research and clinical applications to have devices useful for personalized oncology.

Reversible transition between the epithelial and mesenchymal states are key aspects of carcinoma cell dissemination and the metastatic disease, and thus, characterizing the molecular basis of the epithelial to mesenchymal transition (EMT) is crucial to find druggable targets and more effective therapeutic approaches in cancer. Emerging studies suggest that epigenetic regulators might endorse cancer cells with the cell plasticity required to conduct dynamic changes in cell state during EMT. However, epigenetic mechanisms involved remain mostly unknown. Polycomb Repressive Complexes (PRCs) proteins are well-established epigenetic regulators of development and stem cell differentiation, but their role in different cancer systems is inconsistent and sometimes paradoxical. In this study, we have analysed the role of the PRC2 protein EZH2 in lung carcinoma cells. We found that besides its described role in CDKN2A-dependent cell proliferation, EZH2 upholds the epithelial state of cancer cells by repressing the transcription of hundreds of mesenchymal genes. Chemical inhibition or genetic removal of EZH2 promotes the residence of cancer cells in the mesenchymal state during reversible epithelial-mesenchymal transition. In fitting, analysis of human patient samples and tumour xenograft models indicate that EZH2 is required to efficiently repress mesenchymal genes and facilitate tumour colonization in vivo. Overall, this study discloses a novel role of PRC2 as a master regulator of EMT in carcinoma cells. This finding has important implications for the design of therapies based on EZH2 inhibitors in human cancer patients.

Melanoma is a type of highly malignant and metastatic skin cancer. In situ molecular imaging of endogenous levels of the melanoma biomarker tyrosinase (TYR) may decrease the likelihood of mortality. In this study, we proposed the weakly fluorescent probe 1-(4-(2-(4-(dicyanomethylene)- 4H-chromen-2-yl)vinyl)phenyl)-3-(4-hydroxybenzyl)urea (DCM-HBU), which releases a strong redshifted fluorescent signal after a TYR-mediated oxidation followed by hydrolysis of the urea linkage. The large Stokes shift of the dye is owed to the recovery of the intramolecular charge transfer (ICT) effect. The resulting probe derivate shows a highly ratiometric fluorescence output. Furthermore, the simultaneous excitation by two near-infrared (NIR) photons of the released derivative of dicyanomethylene-4H-pyran (DCM-NH2) fluorophore could avoid the usual drawbacks, such as cellular absorption, autofluorescence, and light scattering, due to an usually short wavelength of the excitation light on biological systems, resulting in images with deeper tissue penetration. In addition, the probe is useful for the quantitative sensing of TYR activity in vivo, as demonstrated in zebrafish larvae. This new ratiometric two-photon NIR fluorescent probe is expected to be useful for the accurate detection of TYR in complex biosystems at greater depths than other one-photon excited fluorescent probes., Ministerio de Ciencia e Innovacion(MCIN/AEI) PID2020-113059GB-C21 RTI2018-101309-B-C22 A-FQM-230-UGR20, Conocimiento y Universidades/Proyecto P18-FR-2470, Consejeria de Economia, Conocimiento, Empresas y Universidad de la Junta de Andalucia (European Regional Development Fund) FPU17/04749, Chair "Doctors Galera-Requena in cancer stem cell research" RH-0019-2020, Consejeria de Salud y Familias (CSyF) of the Regional Government of Andalusia, Spain PAIDI 2021-FEDER funds-POSTDOC_21_638, Plan Andaluz de Investigacion, Desarrollo e Innovacion PID2020-114256RB-I00

Tumours are complex systems with dynamic interactions between tumour cells, nontumour cells, and extracellular components that comprise the tumour microenvironment (TME). The majority of TME’s cells are cancer-associated fibroblasts (CAFs), which are crucial in extracellular matrix (ECM) construction, tumour metabolism, immunology, adaptive chemoresistance, and tumour cell motility. CAF subtypes have been identified based on the expression of protein markers. CAFs may act as promoters or suppressors in tumour cells depending on a variety of factors, including cancer stage. Indeed, CAFs have been shown to promote tumour growth, survival and spread, and secretome changes, but they can also slow tumourigenesis at an early stage through mechanisms that are still poorly understood. Stromal–cancer interactions are governed by a variety of soluble factors that determine the outcome of the tumourigenic process. Cancer cells release factors that enhance the ability of fibroblasts to secrete multiple tumour-promoting chemokines, acting on malignant cells to promote proliferation, migration, and invasion. This crosstalk between CAFs and tumour cells has given new prominence to the stromal cells, from being considered as mere physical support to becoming key players in the tumour process. Here, we focus on the concept of cancer as a non-healing wound and the relevance of chronic inflammation to tumour initiation. In addition, we review CAFs heterogeneous origins and markers together with the potential therapeutic implications of CAFs “re-education” and/or targeting tumour progression inhibition., Consejería de Economía, Conocimiento, Empresas y Universidad de la Junta de Andalucía and European Regional Development Fund (ERDF), ref. P18-FR-2470, Ministry of Science, Innovation and Universities (ref. RTI2018-101309-B-C22), Chair “Doctors Galera-Requena in cancer stem cell research” (CMC-CTS963)

Conventional in vitro cancer models do not accurately reproduce the tumor microenvironment (TME), so three-dimensional (3D)-bioprinting represents an excellent tool to overcome their limitations. Here, two multicellular tri-layered malignant melanoma (MM) models composed by cancer stem cells (CSCs) isolated from a MM established cell line or a primary-patient derived cell line, fibroblasts, mesenchymal stem cells, and endothelial cells, embedded within an agarose-collagen type I hydrogel were bioprinted. Embedded-cells showed high proliferation and metabolic activity, and actively remodeled their TME. MM hydrogels displayed similar rheological properties that skin and were able to support an early onset of vascularization. Besides, MM hydrogels displayed different response to vemurafenib compared with cell cultures, and supported tumorigenesis in murine xenotransplant achieving more mimetic in vivo models. For the first time a tri-layered 3D-bioprinted CSC-based human MM model is developed recreating TME in vitro and in vivo and response to treatment, being useful for precision treatment regimens against MM., Consejería de Salud y Familias de la Junta de Andalucía (Project No. PIN-0224-2019), Consejería de Economía, Conocimiento, Empresas y Universidad de la Junta de Andalucía (FEDER Funds, Projects PY18-FR-2470, B-CTS-230-UGR18, A-CTS-180-UGR20, PYC20 RE 015 UGR and P18-FR-2465), Ministry de Economía y Competitividad, Instituto de Salud Carlos III (FEDER funds, Projects Nos. DTS19/00145 and DTS21/00098), Chair ‘Doctors Galera-Requena in cancer stem cell research’ (CMCCTS963), Universidad de Granada/CBUA., Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020— FEDER funds—Ref: DOC_01574).

The authors have requested that this preprint be removed from Research Square.

European Union [765492 to M.H.]; Spanish Government [AGL2017-88702-C2-2-R to M.H.]; Chair 'Doctors Galera-Requena in cancer stem cell research' (to J.A.M.); Tromsoforskningsstiftelse (TFS) [20 SG BF 'MIRevolution' to B.F.]; Stichting Cancer Center Amsterdam [CCA2021-9-77 to C.G]; TKI-Health Holland ['AQrate' project to C.G. and M.P.]. This publication is part of a project that has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 765492., The NCBI Sequence Read Archive currently hosts microRNA sequencing data for over 800 different species, evidencing the existence of a broad taxonomic distribution in the field of small RNA research. Simultaneously, the number of samples per miRNA-seq study continues to increase resulting in a vast amount of data that requires accurate, fast and user-friendly analysis methods. Since the previous release of sRNAtoolbox in 2019, 55 000 sRNAbench jobs have been submitted which has motivated many improvements in its usability and the scope of the underlying annotation database. With this update, users can upload an unlimited number of samples or import them from Google Drive, Dropbox or URLs. Micro- and small RNA profiling can now be carried out using high-confidence Metazoan and plant specific databases, MirGeneDB and PmiREN respectively, together with genome assemblies and libraries from 441 Ensembl species. The new results page includes straightforward sample annotation to allow downstream differential expression analysis with sRNAde. Unassigned reads can also be explored by means of a new tool that performsmapping to microbial references, which can reveal contamination events or biologically meaningful findings as we describe in the example. sRNAtoolbox is available at: https://arn.ugr.es/srnatoolbox/., European Commission 765492, Spanish Government, European Commission AGL2017-88702-C2-2-R, Chair 'Doctors Galera-Requena in cancer stem cell research', Stichting Cancer Center Amsterdam CCA2021-9-77, Tromsoforskningsstiftelse (TFS) ['MIRevolution'] 20 SG BF, TKI-Health Holland ['AQrate' project]

The physicochemical features of the avian eggshell membrane play an essential role in the process of calcium carbonate deposition during shell mineralization, giving rise to a porous mineralized tissue with remarkable mechanical properties and biological functions. The membrane could be useful by itself or as a bi-dimensional scaffold to build future bone-regenerative materials. This review focuses on the biological, physical, and mechanical properties of the eggshell membrane that could be useful for that purpose. Due to its low cost and wide availability as a waste byproduct of the egg processing industry, repurposing the eggshell membrane for bone bio-material manufacturing fulfills the principles of a circular economy. In addition, eggshell membrane particles have has the potential to be used as bio-ink for 3D printing of tailored implantable scaffolds. Herein, a literature review was conducted to ascertain the degree to which the properties of the eggshell membrane satisfy the requirements for the development of bone scaffolds. In principle, it is biocompatible and non-cytotoxic, and induces proliferation and differentiation of different cell types. Moreover, when implanted in animal models, it elicits a mild inflammatory response and displays characteristics of stability and biodegradability. Furthermore, the eggshell membrane possesses a mechanical viscoelastic behavior comparable to other collagen-based systems. Overall, the biological, physical, and mechanical features of the eggshell membrane, which can be further tuned and improved, make this natural polymer suitable as a basic component for developing new bone graft materials.

Cartilage is a connective tissue which a limited capacity for healing and repairing. In this context, osteoarthritis (OA) disease may be developed with high prevalence in which the use of scaffolds may be a promising treatment. In addition, three-dimensional (3D) bioprinting has become an emerging additive manufacturing technology because of its rapid prototyping capacity and the possibility of creating complex structures. This study is focused on the development of nanocellulose-alginate (NC-Alg) based bioinks for 3D bioprinting for cartilage regeneration to which it is added chondroitin sulfate (CS) and dermatan sulfate (DS). First, rheological properties are evaluated. Then, sterilization effect, biocompatibility, and printability on developed NC-Alg-CS and NC-Alg-DS inks are evaluated. Subsequently, printed scaffolds are characterized. Finally, NC-Alg-CS and NC-Alg-DS inks are loaded with murine D1-MSCs-EPO and cell viability and functionality, as well as the chondrogenic differentiation ability are assessed. Results show that the addition of both CS and DS to the NC-Alg ink improves its characteristics in terms of rheology and cell viability and functionality. Moreover, differentiation to cartilage is promoted on NC-Alg-CS and NC-Alg-DS scaffolds. Therefore, the utilization of MSCs containing NC-Alg-CS and NC-Alg-DS scaffolds may become a feasible tissue engineering approach for cartilage regeneration., The authors thank the Basque Government for granted fellowship to S.Ruiz-Alonso (PRE_2020_2_0143). This study was financially supported bythe Basque Country Government (IT907-16), the Ministerio de Economía,Industria y Competitividad (FEDER funds, project RTC-2016-5451-1). Theyalso wish to thank the intellectual and technical assistance from the ICTS“NANBIOSIS,” more specifically by the Drug Formulation Unit (U10)of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) at the University of Basque Country (UPV/EHU). This research wasalso supported by Fundación Mutua Madrileña (project FMM-AP17196-2019), Consejería de Economía, Conocimiento, Empresas y Universi-dad de la Junta de Andalucía (ERDF funds, projects B-CTS-230-UGR18,SOMM17-6109, and P18-FR-2465), and the Instituto de Salud Carlos III,ERDF funds (DTS19/00145 and DTS21/00098). The authors also thank toSpanish Ministry of Science and Innovation (MICINN) (project PID2020-114086RB-100). The corresponding author information was updated onMarch 14, 2022.

Anti-CD19 chimeric antigen receptor (CAR)-T cells have achieved impressive outcomes for the treatment of relapsed and refractory B-lineage neoplasms.However, important limitations still remain due to severe adverse events (i.e., cytokine release syndrome and neuroinflammation) and relapse of 40%–50%of the treated patients.MostCAR-Tcells are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion, and overstimulation, reducing efficacy and increasing side effects. Here, we show that lentiviral vectors (LVs) expressing the transgene through a WAS gene promoter (AW-LVs) closely mimic the T cell receptor (TCR)/CD3 expression kinetic upon stimulation. These AW-LVs can generate improved CAR-T cells as a consequence of theirmoderate andTCR-like expression profile. Compared with CAR-T cells generated with human elongation factor a (EF1a)-driven-LVs, AW-CAR-T cells exhibited lower tonic signaling, higher proportion of naive and stem cell memory T cells, less exhausted phenotype, and milder secretion of tumor necrosis factor alpha (TNF-a) and interferon (IFN)-ɣ after efficient destruction of CD19+ lymphoma cells, both in vitro and in vivo.Moreover, we also showed their improved efficiency using an in vitro CD19+ pancreatic tumor model. We finally demonstrated the feasibility of large-scale manufacturing ofAW-CAR-T cells in good manufacturing practice (GMP)-like conditions. Based on these data, we propose the use of AW-LVs for the generation of improved CAR-T products., Spanish ISCIII Health Research Fund, European Commission PI15/02015 PI18/00337 PI21/00298 RD21/0017/0004 PI18/00330 PI17/00672, CSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) for Andalusia 2016000073391-TRA 2016000073332-TRA PI-57069 PA IDI-Bio326 CARTPI-0001-201 PECART-0031-2020 Red RANTECAR CAR-T 2019 00400200101918 PLEC2021-008094 PI-0014-2016 PEER-0286-2019, Spanish Government PLEC2021-008094 00123009/SNEO-20191072, Nicolas Monardes contracts from regional Ministry of Health 0006/2018 C2-0002-2019, German Research Foundation (DFG) FPU16/05467 FPU17/02268 FPU17/04327 MCI DIN2018-010180, Fundacion Andaluza Progreso y Salud, German Research Foundation (DFG) PEJ-2018-001760-A, Junta de Andalucia PE-0223-2018, Biomedicine Programme of the University of Granada (Spain)

The increase in cancer incidences shows that there is a need to better understand tumour heterogeneity to achieve efficient treatments. Interestingly, there are several common features among almost all types of cancers, with chronic inflammation induction and deaminase dysfunctions singled out. Deaminases are a family of enzymes with nucleotide-editing capacity, which are classified into two main groups: DNA-based and RNA-based. Remarkably, a close relationship between inflammation and the dysregulation of these molecules has been widely documented, which may explain the characteristic intratumor heterogeneity, both at DNA and transcriptional levels. Indeed, heterogeneity in cancer makes it difficult to establish a unique tumour progression model. Currently, there are three main cancer models—stochastic, hierarchic, and dynamic—although there is no consensus on which one better resembles cancer biology because they are usually overly simplified. Here, to accurately explain tumour progression, we propose interactions among chronic inflammation, deaminases dysregulation, intratumor genetic heterogeneity, cancer phenotypic plasticity, and even the previously proposed appearance of cancer stem-like cell populations in the edges of advanced solid tumour masses (instead of being the cells of origin of primary malignancies). The new tumour development model proposed in this study does not contradict previously accepted models and it may open up a window to interesting therapeutic approaches.

Osteoarthritis is a disease with a great socioeconomic impact and mainly affects articular cartilage, a tissue with reduced self-healing capacity. In this work, 3D printed 1,4 butanediol thermoplastic polyurethane (b-TPUe) scaffolds are functionalized and infrapatellar mesenchymal stem cells are used as the cellular source. Since b-TPUe is a biomaterial with mechanical properties similar to cartilage, but it does not provide the desired environment for cell adhesion, scaffolds are functionalized with two methods, one based on collagen type I and the other in 1-pyrenebutiric acid (PBA) as principal components. Alamar Blue and confocal assays display that PBA functionalized scaffolds support higher cell adhesion and proliferation for the first 21 days. However, collagen type I functionalization induces higher proliferation rates and similar cell viability than the PBA method. Further, both functionalization methods induce extracellular matrix synthesis, and the presence of chondrogenic markers (Sox9, Col2a, and Acan). Finally, SEM images probe that functionalized 3D printed scaffolds present much better cell/biomaterial interactions than controls and confirm early chondrogenesis. These results indicate that the two methods of functionalization in the highly hydrophobic b-TPUe enhance the cell-biomaterial interactions and the improvement in the chondro-inductive properties, which have great potential for application in cartilage tissue engineering.

Osteochondral (OC) lesions are a major cause of chronic musculoskeletal pain and functional disability, which reduces the quality of life of the patients and entails high costs to the society. Currently, there are no effective treatments, so in vitro and in vivo disease models are critically important to obtain knowledge about the causes and to develop effective treatments for OC injuries. In vitro models are essential to clarify the causes of the disease and the subsequent design of the first barrier to test potential therapeutics. On the other hand, in vivo models are anatomically more similar to humans allowing to reproduce the pattern and progression of the lesion in a controlled scene and offering the opportunity to study the symptoms and responses to new treatments. Moreover, in vivo models are the most suitable preclinical model, being a fundamental and a mandatory step to ensure the successful transfer to clinical trials. Both in vitro and in vitro models have a number of advantages and limitation, and the choice of the most appropriate model for each study depends on many factors, such as the purpose of the study, handling or the ease to obtain, and cost, among others. In this chapter, we present the main in vitro and in vivo OC disease models that have been used over the years in the study of origin, progress, and treatment approaches of OC defects.

In this study, the synthesis and characterization of lipid nanocapsules coated with folate–chitosan conjugates at varying folate concentrations are reported; these nanocapsules have a potential application as anticancer drug carriers. The main goal of this study was to evaluate (a) the colloidal stability of the particles and (b) their cell targeting. A classical colloidal characterization of the nanocapsules was carried out by analyzing size, electrokinetic charge, and stability in different saline solutions, including cell culture media. At neutral pH, the stability was improved by the presence of folate due to electrical interactions. In addition, folate modulated the hydrophilic/phobic nature of the surface, which became critical to keep the systems stable (or not) under physiological saline conditions due to the action of short-range repulsive hydration forces. The cellular uptake of our nanocapsules was evaluated by working with four tumor cell lines. Both fluorescent analyses with particles colored by Nile Red, and antitumor activity of our systems loaded with docetaxel, demonstrated that the folate-mediated internalization of the particles in the cancer cells was improved when the nanocapsules were coated by folate–chitosan conjugates.

Progress in nanotechnology has enabled us to open many new fronts in biomedical research by exploiting the peculiar properties of materials at the nanoscale. The thermal sensitivity of certain materials is a highly valuable property because it can be exploited in many promising applications, such as thermo-sensitive drug or gene delivery systems, thermotherapy, thermal biosensors, imaging, and diagnosis. This review focuses on recent advances in thermo-sensitive nanomaterials of interest in biomedical applications. We provide an overview of the different kinds of thermoresponsive nanomaterials, discussing their potential and the physical mechanisms behind their thermal response. We thoroughly review their applications in biomedicine and finally discuss the current challenges and future perspectives of thermal therapies., This research was supported by the Fundación Mutua Madrileña (project FMM-AP16683-2017), Consejería de Salud Junta de Andalucía (PI-0089-2017), Instituto de Salud Carlos III (FEDER funds PIE16/00045), MINECO MAT2015-63644-C2-R, MAT 2016-78778-R, PCIN-2015-051 projects (Spain), European Regional Development Fund (ERDF) and from the Chair “Doctors Galera-Requena in cancer stem cell research”.

As leads we took several benzo-fused seven- and six-membered scaffolds linked to the pyrimidine or purine moieties with notable anti-proliferative activity against human breast, colon and melanoma cancerous cell lines. We then decided to maintain the double-ringed nitrogenous bases and change the other components to the ethyl acetate moiety. This way six purine and two 5-fluorouracil derivatives were obtained and evaluated against the MCF-7, HCT-116, A-375 and G-361 cancer cell lines. Two QSARs are obtained between the anti-proliferative IC 50 values for compounds 26–33 and the c log P against the melanoma cell lines A-375 and G-361. Our results show that two of the analogues [ethyl 2-(2,6-dichloro-9 H - or 7 H -purine-9- or 7-yl)acetates ( 30 and 33 , respectively)] are potent cytotoxic agents against all the tumour cell lines assayed, showing single-digit micromolar IC 50 values. This exemplifies the potential of our previously reported purine compounds to qualify as lead structures for medicinal chemistry campaigns, affording simplified analogues easy to synthesize and with a noteworthy bioactivity. The selective activity of 30 and 33 against the melanoma cell line A-375, via apoptosis, supposes a great advantage for a future therapeutic use.

Preinfarction angina, a possible form of ischaemic preconditioning, improves the prognosis in patients who experience a major ischaemic event; though the associated pathophysiology is not yet fully understood. The aim of this study was to determine the possible involvement of endothelial progenitor cells (EPC), the vascular endothelial growth factor (VEGF) and the hepatocyte growth factor (HGF) in the development of preinfarction angina.We studied 41 patients (60·5 ± 12 years; 34% women) and 14 healthy controls; 43·9% of the patients had preinfarction angina. No differences were found in the baseline characteristics of the two groups. Although the EPC, VEGF and HGF were raised as compared with the control group, no significant differences were found according to the presence or absence of preinfarction angina in the levels of EPC (baseline, P = 0·25; day 3, P = 0·11; day 7, P = 0·32), VEGF (baseline, P = 0·96; day 3, P = 0·06; day 7, P = 0·57) or HGF (baseline, P = 0·18; day 3, P = 1; day 7, P = 0·86). An association was seen in the patients who had preinfarction angina between the EPC levels at baseline and on days 3 and 7 and the HGF on admission with the time from the angina to the STEMI (β = -0·070; β = -0·066; β = -0·081; β = -80·16; P0·05), showing a reduction in the level of EPC cells for each hour passed since the event.No differences were found in the release kinetics of EPC, VEGF or HGF after a first infarction according to whether the patients had angina during the week before the infarction.

Following an acute myocardial infarction (AMI), bone-marrow derived endothelial progenitor cells (EPC) are mobilised into the peripheral blood. Our aim was to examine the factors influencing this spontaneous cell mobilisation.In this study we analysed 47 patients with extensive AMI (left ventricular ejection fraction [LVEF]50% by echocardiography during the first week post-AMI); we studied the peripheral blood EPC populations expressing CD133(+), CD34(+), KDR(+), CXCR4(+), as well as the cytokines VEGF (vascular endothelial growth factor), SDF-1 (stromal cell-derived factor 1) and TSP-1 (thrombospondin 1), measured on day 5±2.5 after AMI.The extension of AMI (CPK peak) correlated with the number of CD133(+) mobilised cells: (r=0.40; P=.011). Patients who did not receive perfusion during the acute phase (34%) had more CD34(+)CXCR4(+) cells with a median (interquartile ranges) of 2,401 (498-7,004) vs. 999 (100-1,600), P=.048, and strong correlations between VEGF and CD133(+)CD34(+)KDR(+) (r=.84; P.01) and SDF-1 and CD34(+)CXCR4(+) (r=.67; P.01), and between these 2 cytokines (r=.57; P=.01). In the reperfused patients, the correlation between VEGF and CD133(+)CD34(+)KDR(+) was lower (r=.38; P=.03) and the correlation between SDF-1 and CD34(+)CXCR4(+) and VEGF disappeared. Multivariate analysis showed that a VEGF7pg/mL (P.01) predicted the mobilisation of CD133(+)CD34(+)KDR(+), whereas hypertension showed a trend (P=.055). Diabetes (P=.045) predicted the number of CD34(+)CXCR4(+), with reperfusion treatment showing a trend in this subpopulation (P=.054).Mobilisation of progenitor cells after AMI is influenced by factors such as diabetes and the cytokine VEGF. Hypertension and reperfusion therapy during the acute phase also tend to influence the cell response.

Differentiation is a complex multistep process of cell specialization that begins with the installation of a genetic programme, named determination, specific for a cell lineage. Development of the differentiation programme includes the cell-type specific silencing of some genes and the expression of other genes, that regulate the biological functions associated with the cellular type and that distinguish the specialized cells. Terminal differentiation is the end stage of this process where the cells irreversibly lose their proliferative capacity and which represents a form of negative control of growing. Regulating molecules interact to produce the correct balance between cellular multiplication and differentiation during embryogenesis and the normal behaviour of an adult. Cancer is a process in which changes in regulating circuits are produced, such as proliferation control, the balance between cellular survival and programmed cellular death (apoptosis), the communication with neighbouring cells and with the extracellular matrix, angiogenesis, and finally, the migration of the tumoural cell, the invasion and metastasic dissemination. This process implies the progressive development of a more malign phenotype with an increase of genetic alterations involving genes at several levels of expression during long periods of time. These genetic changes uncouple the normal balance between multiplication and cellular differentiation with an increase in the rate of proliferating cells. Classic chemotherapeutical agents have been very important; nevertheless, as the mechanism of action of these drugs depends on the cytodestruction of the neoplastic cells, their beneficial effects are normally accompanied by a notable morbidity, cytotoxicity and multidrug resistance. The knowledge of the mechanisms involved in differentiation and malignant transformation has allowed the search of alternative routes for antitumoural therapy that does not imply cellular death. Differentiation therapy focuses on the development and use of specific agents designed to selectively attract the terminal differentiation process, making the elimination of tumoural cells feasible together with the establishment of normal cellular homeostasis.

Cadmium is a heavy metal dangerous for the environment and for the human health, with well shown carcinogenic potentiality. Many studies have related the professional exposure of the Cadmium with human pulmonary, prostatic and renal tumors, and it would be a role also in the tumors of the liver, of the hemopoietic system, of the bladder and of the stomach. The aim of the current study is to examine in normal and neoplastic fibroblasts culture cells the modifications induced by the Cadmium at cellular level, in particular on the cytoskeleton, responsible not only of the intracellular transport of vesicles and cell organules, but also of their positioning and of the cellular integrity. Two fibroblastic cellular strains, normal (FG) and neoplastic (SGS/3A), have been incubated in a 5 microM Cadmium acetate added medium for 1, 8, 24 hours and studied by indirect immunofluorescence methods, particularly for the following proteins: Actin, Tubulin and Vimentin. The observations show in normal and neoplastic fibroblasts comparables modifications and anomalies of cytoskeletal shape. In both the cases the cellular morphology suffers drastic modifications, gradually evolving through intermediary shapes: from triangular and spindle-shaped in the normal fibroblasts to irregular, star-shaped, and globular in the neoplastic ones. The Cadmium action on the morphology of the normal and tumoral cells changes according to the time of incubation, producing structural alterations of the cytoskeletal. The modifications that start to be observable at the first hour of incubation are more evident after the eighth hour of exposure, reaching the maximum expression at the twenty-fourth hour, often with reduction of the total volume of the cells and loss of their ability to adhere to the substratum. Such modifications can be related to great alterations of the cellular membrane, producing the change of shape and the progressive partial separation from the substratum. The intermediary filaments seem to be less sensitive, from a morphological but not functional point of view, to the action of the Cadmium in comparison to the Actin and the microtubules that, on the contrary, seem to lose their proper morphological characteristics.

Convenient synthesis of 1-[[3-(2-hydroxyethylhetero)-1-alkoxy]alkyl]-5-fluorouracils 3-8 was accomplished via the use of tin (IV) chloride, capable of a 1,4-chelation on alkoxy-1,4-diheteroepanes. Increasing the reaction time led to 5-FU seven-membered nucleoside analogues which could be considered as upper isosteres of the effective antitumour agent Ftorafur. Using 1-[[3-(2-hydroxyethoxy)-1-alkoxy]propyl]-5-fluorouracil as a parent drug, several chemical modifications on the acyclic moiety were made with the aim of obtaining new compounds showing significant antiproliferative activity in rhabdomyosarcoma (RD) cells. 14 treatment in vitro caused time- and dose-dependent growth inhibition on RD cells. Interestingly, when they were treated with doses of 35 microM and 140 microM of 14 for 6 days, they showed morphological and phetotypic differentiation with increased expression of desmin, alpha-actinin and tropomyosin. We suggest a potential role for differentiation therapy as a therapeutic approach to the treatment of rhabdomyosarcoma.

New therapeutic strategies are required to overcome the limitations of conventional breast cancer treatment. Suicide gene therapy offers a potential approach to this type of tumour, since systems based on the use of prodrugs may present some drawbacks related to toxicity, drug release and bioavailability. The gef gene has cell-killing functions in Escherichia coli and does not depend on the use of a prodrug for its action, making it an attractive target for suicide gene therapy. We created a gef-overexpressing human breast cancer cell line (MCF-7TG) by transfecting the gef gene under the control of a pMAMneo promotor. Dexamethasone-induction of gef gene expression in MCF-7TG cells produced a significant decrease in Ki-67 expression, which is a known proliferation marker. In addition, annexin-V-FITC and propidium iodide assays showed the presence of apoptotic cell death, which was confirmed by scanning electron microscopy. The most significant finding was the presence of "craters" in the cell membrane, as previously described in other apoptotic breast cancer cells. These results demonstrate the ability of the gef gene to down regulate Ki-67 expression and induce apoptosis in a breast cancer cell line, suggesting its potential application as a new gene therapy strategy for this type of tumor.

An abnormal HLA expression has been detected in some tumors including rhabdomyosarcoma (RMS). Classical cytotoxic treatment of these tumors, the most common childhood soft tissue malignancy, may induce multidrug resistance (MDR) associated with the expression of a 170-kDa membrane-associated glycoprotein (P-glycoprotein). In order to analyse the connection between modulation of HLA expression and the development of the MDR phenotype mediated by P-glycoprotein in RMS, we used three resistant RMS cell lines; two of these resistant cell lines (TE.32.7.DAC and RD-DAC) were established by in vitro exposure to actinomycin D, a drug of choice in the treatment of RMS; the resistant RMS- GR cell line was established from an embryonal RMS tumor after polychemotherapy. Our results showed that all the resistant cell lines showed a significant increase in the expression of HLA class I surface antigens in comparison to drug-sensitive cells. Blockade of P-glycoprotein with verapamil led to a decrease in HLA class I expression in RMS resistant cell lines. However, no modulation of HLA class II expression was observed in any of the three analyzed cell lines. These findings support the hypothesis that the development of resistance mediated by mdr 1/P-glycoprotein, directly influences the expression of HLA class I in RMS cells, inducing to upregulation. This effect may be relevant to the application in RMS of immunotherapy against tumor-associated antigens presented by HLA class I molecules.