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Belgrade is the most popular tourist destination in Serbia, whose tourism industry is rapidly recovering from the turbulent events of the 1990s. However, Belgrade's destination managers yet need to use to a greater extent its competitive advantages which would improve the city's ability to compete with more prominent cities in the international market. These managerial efforts should be based on relevant image assessments. The aim of this research is to offer a comprehensive insight into Belgrade's destination image by combining quantitative and qualitative data collection methods in order to identify the crucial components of its cognitive and affective images. Additionally, this study examines the influence of various tourists’ socio-demographic characteristics on their perceptions of destination image, providing both theoretical and marketing implications. Cultural attractions, nightlife, gastronomy and history were identified in the qualitative study as the most important elements of Belgrade’s cognitive image, while the most common affective associations were related to it being vibrant, authentic and relaxing. In the quantitative study, six factors of Belgrade’s cognitive image were identified - Tourist Attractions, Services and Fun, Environment, Accessibility, Local Residents, and River Banks and Green Areas. Regarding the affective component, it was established that Belgrade was primarily perceived as pleasant, but also as exciting, cheerful and relaxing. Hypotheses testing indicated that tourists’ affective responses towards Belgrade varied in relation to their gender and geographical origin, while some of their cognitive perceptions differed in relation to their gender, age, education and employment. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 176017: Problems and tendencies of development of geospatial systems of the Republic of Serbia and Grant no. 176008: Development programs of villages revitalization in Serbia]

Breast cancer comprises a substantial proportion of cancer diagnoses in women and is a primary cause of cancer-related mortality. While hormone-responsive cases generally have a favorable prognosis, the aggressive nature of triple-negative breast cancer presents challenges, with intrinsic resistance to established treatments being a persistent issue. The complexity intensifies with the emergence of acquired resistance, further complicating the management of breast cancer. Epigenetic changes, encompassing DNA methylation, histone and RNA modifications, and non-coding RNAs, are acknowledged as crucial contributors to the heterogeneity of breast cancer. The unique epigenetic landscape harbored by each cellular component within the tumor microenvironment (TME) adds great diversity to the intricate regulations which influence therapeutic responses. The TME, a sophisticated ecosystem of cellular and non-cellular elements interacting with tumor cells, establishes an immunosuppressive microenvironment and fuels processes such as tumor growth, angiogenesis, and extracellular matrix remodeling. These factors contribute to challenging conditions in cancer treatment by fostering a hypoxic environment, inducing metabolic stress, and creating physical barriers to drug delivery. This article delves into the complex connections between breast cancer treatment response, underlying epigenetic changes, and vital interactions within the TME. To restore sensitivity to treatment, it emphasizes the need for combination therapies considering epigenetic changes specific to individual members of the TME. Recognizing the pivotal role of epigenetics in drug resistance and comprehending the specificities of breast TME is essential for devising more effective therapeutic strategies. The development of reliable biomarkers for patient stratification will facilitate tailored and precise treatment approaches., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

3D organoids are widely used as tractable in vitro models capable of elucidating aspects of human development and disease. However, the manual and low-throughput culture methods, coupled with a low reproducibility and geometric heterogeneity, restrict the scope and application of organoid research. Combining expertise from stem cell biology and bioengineering offers a promising approach to address some of these limitations. Here, melt electrospinning writing is used to generate tuneable grid scaffolds that can guide the self-organization of pluripotent stem cells into patterned arrays of embryoid bodies. Grid geometry is shown to be a key determinant of stem cell self-organization, guiding the position and size of emerging lumens via curvature-controlled tissue growth. Two distinct methods for culturing scaffold-grown embryoid bodies into either interconnected or spatially discrete cerebral organoids are reported. These scaffolds provide a high-throughput method to generate, culture, and analyze large numbers of organoids, substantially reducing the time investment and manual labor involved in conventional methods of organoid culture. It is anticipated that this methodological development will open up new opportunities for guiding pluripotent stem cell culture, studying lumenogenesis, and generating large numbers of uniform organoids for high-throughput screening., (© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers worldwide, primarily due to its robust desmoplastic stroma and immunosuppressive tumor microenvironment (TME), which facilitate tumor progression and metastasis. In addition, fibrous tissue leads to sparse vasculature, high interstitial fluid pressure, and hypoxia, thereby hindering effective systemic drug delivery and immune cell infiltration. Thus, remodeling the TME to enhance tumor perfusion, increase drug retention, and reverse immunosuppression has become a key therapeutic strategy. In recent years, targeting epigenetic pathways has emerged as a promising approach to overcome tumor immunosuppression and cancer progression. Moreover, the progress in nanotechnology has provided new opportunities for enhancing the efficacy of conventional and epigenetic drugs. Nano-based drug delivery systems (NDDSs) offer several advantages, including improved drug pharmacokinetics, enhanced tumor penetration, and reduced systemic toxicity. Smart NDDSs enable precise targeting of stromal components and augment the effectiveness of immunotherapy through multiple drug delivery options. This review offers an overview of the latest nano-based approaches developed to achieve superior therapeutic efficacy and overcome drug resistance. We specifically focus on the TME and epigenetic-targeted therapies in the context of PDAC, discussing the advantages and limitations of current strategies while highlighting promising new developments. By emphasizing the immense potential of NDDSs in improving therapeutic outcomes in PDAC, our review paves the way for future research in this rapidly evolving field., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Recent advances in the three-dimensional (3D) cancer models give rise to a plethora of new possibilities in the development of anti-cancer drug therapies and bring us closer to personalized medicine. Three-dimensional models are undoubtedly more authentic than traditional two-dimensional (2D) cell cultures. Nowadays, they are becoming preferentially used in most cancer research fields due to their more accurate biomimetic characteristics. On the contrary, they still lack the cellular and matrix complexity of the native tumor microenvironment (TME). This review focuses on the description of existing 3D models, the incorporation of TME and fluidics into these models, and their perspective in the future research. It is clear that such an improvement would need not only biological but also technical progress. Therefore, the modern approach to anti-cancer drug discovery should involve various fields., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Pancreatic neuroendocrine neoplasms (pNENs) are rare cancers with broad challenges for their management. The main clinical obstacles are the high rate of patients diagnosed at advanced stages, lack of prognostic markers for early detection of disease recurrence in resected patients, significant limitations in identifying those who will benefit from adjuvant therapy, and timely recognition of treatment response. Therefore, the discovery of new prognostic and predictive markers is necessary for patient stratification and clinical management. Liquid biopsy, which has revolutionized the field of clinical oncology, is extremely under-investigated in pNENs. This review highlights its potential and the recent advances in related technologies, as candidates for the delivery of the new tools that can help to refine pNEN diagnosis and to personalize treatment. In addition, the opportunities and limitations of available preclinical research models with regard to biomarker research are discussed in light of pNEN clinical needs., Competing Interests: Conflict of interest statement All authors declare no conflict of interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

3D printing is a powerful manufacturing technology for shaping materials into complex structures. While the palette of printable materials continues to expand, the rheological and chemical requisites for printing are not always easy to fulfill. Here, a universal manufacturing platform is reported for shaping materials into intricate geometries without the need for their printability, but instead using light-based printed salt structures as leachable molds. The salt structures are printed using photocurable resins loaded with NaCl particles. The printing, debinding, and sintering steps involved in the process are systematically investigated to identify ink formulations enabling the preparation of crack-free salt templates. The experiments reveal that the formation of a load-bearing network of salt particles is essential to prevent cracking of the mold during the process. By infiltrating the sintered salt molds and leaching the template in water, complex-shaped architectures are created from diverse compositions such as biomedical silicone, chocolate, light metals, degradable elastomers, and fiber composites, thus demonstrating the universal, cost-effective, and sustainable nature of this new manufacturing platform., (© 2022 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

Thermoresponsive polymers with the appropriate structure form physical networks upon changes in temperature, and they find utility in formulation science, tissue engineering, and drug delivery. Here, we report a cost-effective biocompatible alternative, namely OEGMA300 15 - b -BuMA 26 - b -DEGMA 13 , which forms gels at low concentrations (as low as 2% w/w); OEGMA300, BuMA, and DEGMA stand for oligo(ethylene glycol) methyl ether methacrylate (MM = 300 g mol -1 ), n -butyl methacrylate, and di(ethylene glycol) methyl ether methacrylate, respectively. This polymer is investigated in depth and is compared to its commercially available counterpart, Poloxamer P407 (Pluronic F127). To elucidate the differences in their macroscale gelling behavior, we investigate their nanoscale self-assembly by means of small-angle neutron scattering and simultaneously recording their rheological properties. Two different gelation mechanisms are revealed. The triblock copolymer inherently forms elongated micelles, whose length increases by temperature to form worm-like micelles, thus promoting gelation. In contrast, Pluronic F127's micellization is temperature-driven, and its gelation is attributed to the close packing of the micelles. The gel structure is analyzed through cryogenic scanning and transmission electron microscopy. Ex vivo gelation study upon intracameral injections demonstrates excellent potential for its application to improve drug residence in the eye., Competing Interests: The authors declare the following competing financial interest(s): We (APC and TKG) have published a patent on this work that covers the novel combination of comonomers, i.e., the chemistry of the polymers., (© 2022 American Chemical Society.)

Acquired drug resistance and metastasis in breast cancer (BC) are coupled with epigenetic deregulation of gene expression. Epigenetic drugs, aiming to reverse these aberrant transcriptional patterns and sensitize cancer cells to other therapies, provide a new treatment strategy for drug-resistant tumors. Here we investigated the ability of DNA methyltransferase (DNMT) inhibitor decitabine (DAC) to increase the sensitivity of BC cells to anthracycline antibiotic doxorubicin (DOX). Three cell lines representing different molecular BC subtypes, JIMT-1, MDA-MB-231 and T-47D, were used to evaluate the synergy of sequential DAC + DOX treatment in vitro. The cytotoxicity, genotoxicity, apoptosis, and migration capacity were tested in 2D and 3D cultures. Moreover, genome-wide DNA methylation and transcriptomic analyses were employed to understand the differences underlying DAC responsiveness. The ability of DAC to sensitize trastuzumab-resistant HER2-positive JIMT-1 cells to DOX was examined in vivo in an orthotopic xenograft mouse model. DAC and DOX synergistic effect was identified in all tested cell lines, with JIMT-1 cells being most sensitive to DAC. Based on the whole-genome data, we assume that the aggressive behavior of JIMT-1 cells can be related to the enrichment of epithelial-to-mesenchymal transition and stemness-associated pathways in this cell line. The four-week DAC + DOX sequential administration significantly reduced the tumor growth, DNMT1 expression, and global DNA methylation in xenograft tissues. The efficacy of combination therapy was comparable to effect of pegylated liposomal DOX, used exclusively for the treatment of metastatic BC. This work demonstrates the potential of epigenetic drugs to modulate cancer cells' sensitivity to other forms of anticancer therapy., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

A vaccine antigen, when launched as DNA or RNA, can be presented in various forms, including intracellular, secreted, membrane-bound, or on extracellular vesicles (EVs). Whether an antigen in one or more of these forms is superior in immune induction remains unclear. In this study, we used GFP as a model antigen and first compared the EV-loading efficiency of transmembrane domains (TMs) from various viral glycoproteins, and then investigated whether EV-bound GFP (EV-GFP) would enhance immune induction. Our data showed that GFP fused to viral TMs was successfully loaded onto the surface of EVs. In addition, GFP-bound EVs were predominantly associated with the exosome marker CD81. Immunogenicity study with EV-GFP-producing plasmids in mice demonstrated that antigen-specific IgG and IgA were significantly increased in EV-GFP groups, compared to soluble and intracellular GFP groups. Similarly, GFP-specific T cell response-related cytokines produced by antigen-stimulated splenocytes were also enhanced in mice immunized with EV-GFP constructs. Immunogenicity study with purified soluble GFP and GFP EVs further confirmed the immune enhancement property of EV-GFP in mice. In vitro uptake assays indicated that EV-GFP was more efficiently taken up than soluble GFP by mouse splenocytes and such uptake was B cell preferential. Taken together, our data indicate that viral TMs can efficiently load antigens onto the EV surface, and that EV-bound antigen enhances both humoral and cell-mediated antigen-specific responses., (© 2022 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Background: Chemotherapy remains a standard treatment option for breast cancer despite its toxic effects to normal tissues. However, the long-lasting effects of chemotherapy on non-malignant cells may influence tumor cell behavior and response to treatment. Here, we have analyzed the effects of doxorubicin (DOX) and paclitaxel (PAC), commonly used chemotherapeutic agents, on the survival and cellular functions of mesenchymal stromal cells (MSC), which comprise an important part of breast tumor microenvironment., Methods: Chemotherapy-exposed MSC (DOX-MSC, PAC-MSC) were co-cultured with three breast cancer cell (BCC) lines differing in molecular characteristics to study chemotherapy-triggered changes in stromal compartment of the breast tissue and its relevance to tumor progression in vitro and in vivo. Conditioned media from co-cultured cells were used to determine the cytokine content. Mixture of BCC and exposed or unexposed MSC were subcutaneously injected into the immunodeficient SCID/Beige mice to analyze invasion into the surrounding tissue and possible metastases. The same mixtures of cells were applied on the chorioallantoic membrane to study angiogenic potential., Results: Therapy-educated MSC differed in cytokine production compared to un-exposed MSC and influenced proliferation and secretory phenotype of tumor cells in co-culture. Histochemical tumor xenograft analysis revealed increased invasive potential of tumor cells co-injected with DOX-MSC or PAC-MSC and also the presence of nerve fiber infiltration in tumors. Chemotherapy-exposed MSC have also influenced angiogenic potential in the model of chorioallantoic membrane., Conclusions: Data presented in this study suggest that neoadjuvant chemotherapy could possibly alter otherwise healthy stroma in breast tissue into a hostile tumor-promoting and metastasis favoring niche. Understanding of the tumor microenvironment and its complex net of signals brings us closer to the ability to recognize the mechanisms that prevent failure of standard therapy and accomplish the curative purpose., (© 2021. The Author(s).)

Taking into account the factors of high incidence rate, prevalence and mortality, breast cancer represents a crucial social and economic burden. Most cases of breast cancer develop as a consequence of somatic mutations accumulating in mammary epithelial cells throughout lifetime and approximately 5-10% can be ascribed to monogenic predispositions. Even though the role of genetic predispositions in breast cancer is well described in the context of genetics, very little is known about the role of the microenvironment carrying the same aberrant cells impaired by the germline mutation in the breast cancer development and progression. Based on the clinical observations, carcinomas carrying mutations in hereditary tumor-suppressor genes involved in maintaining genome integrity such as BRCA1/2 have worse prognosis and aggressive behavior. One of the mechanisms clarifying the aggressive nature of BRCA -associated tumors implies alterations within the surrounding adipose tissue itself. The objective of this review is to look at the role of BRCA1/2 mutations in the context of breast tumor microenvironment and plausible mechanisms by which it contributes to the aggressive behavior of the tumor cells.

Programmed death ligand 1 (PD-L1) overexpression has been associated with poor clinical outcomes in several human cancers whose increased malignant behaviour might be related to PD-L1 mediated systemic immunological tolerance. This study aims to verify if circulating cytokines may serve as a proxy for non-invasive identification of sensitive prognostic biomarkers reflecting tumour and its microenvironment. Immunohistochemistry was used to measure PD-L1 expression in tumour tissue sections of 148 chemonaïve breast cancer (BC) patients. The panel of 51 cytokines was analysed using multiplex bead arrays. High PD-L1 expression in tumours was associated with shorter progression-free survival (HR 3.25; 95% CI 1.39-7.61; P = 0.006) and low circulating levels of three multifunctional molecules; VEGF, TNF-β and IL-15 (P = 0.001). In multivariate analysis, patients with low VEGF had 4.6-fold increased risk of PD-L1 overexpression (P = 0.008), present in 76.5% of patients with all these three cytokines below the median (vs. 35.6% among the others; P = 0.002). The area under the curve value of 0.722 (95% CI 0.59-0.85; P = 0.004) shows that this combination of cytokines has a moderate ability to discriminate between PD-L1 high vs. PD-L1 low patients. Plasma cytokines, therefore, could serve as potential non-invasive biomarkers for the identification of high-risk BC cases.

Circulating tumor cells (CTCs) and the immune infiltration of tumors are closely related to clinical outcomes. This study aimed to verify the influence of stromal lymphocyte infiltration and the immune context of tumor microenvironment on the hematogenous spread and prognosis of 282 chemotherapy naïve primary BC patients. To detect the presence of mesenchymal CTCs, RNA extracted from CD45-depleted peripheral blood was interrogated for the expression of mesenchymal gene transcripts. Tumor-infiltrating lymphocytes (TILs) were detected in the stromal areas by immunohistochemistry, using CD3, CD8, and CD45RO antibodies. The concentrations of 51 plasma cytokines were measured by multiplex bead arrays. TILs infiltration in mesenchymal CTC-positive patients significantly decreased their progression-free survival (HR = 4.88, 95% CI 2.30-10.37, p < 0.001 for CD3 high ; HR = 6.17, 95% CI 2.75-13.80, p < 0.001 for CD8 high ; HR = 6.93, 95% CI 2.86-16.81, p < 0.001 for CD45RO high ). Moreover, the combination of elevated plasma concentrations of transforming growth factor beta-3 (cut-off 662 pg/mL), decreased monocyte chemotactic protein-3 (cut-off 52.5 pg/mL) and interleukin-15 (cut-off 17.1 pg/mL) significantly increased the risk of disease recurrence (HR = 4.838, 95% CI 2.048-11.427, p < 0.001). Our results suggest a strong impact of the immune tumor microenvironment on BC progression, especially through influencing the dissemination and survival of more aggressive, mesenchymal CTC subtypes.

Over the last decade, demand has increased for developing new, alternative materials in pediatric trauma care to overcome the disadvantages associated with conventional implant materials. Magnesium (Mg)-based alloys seem to adequately fulfill the vision of a homogeneously resorbable, biocompatible, load-bearing and functionally supportive implant. The aim of the present study is to introduce the high-strength, lean alloy Mg‒0.45Zn‒0.45Ca, in wt% (ZX00), and for the first time investigate the clinical applicability of screw osteosynthesis using this alloy that contains no rare-earth elements. The alloy was applied in a growing sheep model with osteotomized bone (simulating a fracture) and compared to a non-osteotomy control group regarding degradation behavior and fracture healing. The alloy exhibits an ultimate tensile strength of 285.7 ± 3.1 MPa, an elongation at fracture of 18.2 ± 2.1%, and a reduced in vitro degradation rate compared to alloys containing higher amounts of Zn. In vivo, no significant difference between the osteotomized bone and the control group was found regarding the change in screw volume over implantation time. Therefore, it can be concluded that the fracture healing process, including its effects on the surrounding area, has no significant influence on degradation behavior. There was also no negative influence from hydrogen-gas formation on fracture healing. Despite the proximal and distal screws showing chronologically different gas release, the osteotomy showed complete consolidation. STATEMENT OF SIGNIFICANCE: Conventional implants involve several disadvantages in pediatric trauma care. Magnesium-based alloys seem to overcome these issues as discussed in the recent literature. This study evaluates the clinical applicability of high-strength lean Mg‒0.45Zn‒0.45Ca (ZX00) screws in a growing-sheep model. Two groups, one including a simulated fracture and one group without fracture, underwent implantation of the alloy and were compared to each other. No significant difference regarding screw volume was observed between the groups. There was no negative influence of hydrogen-gas formation on fracture healing and a complete fracture consolidation was found after 12 weeks for all animals investigated., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

A correlation between circulating tumor cells (CTCs) and monocytes in metastatic breast cancer (BC), where CTCs and monocyte-to-lymphocyte ratio (MLR) were predictors of overall survival (OS), was recently shown. Herein, we aimed to assess the association between CTCs and the complete blood count (CBC)-derived inflammation-based scores in 284 primary BC patients. CTCs were determined in CD45-depleted peripheral blood mononuclear cells by real time-PCR. This method allowed us to detect a subset of CTCs with an epithelial-to-mesenchymal transition phenotype (CTC EMT), previously associated with inferior outcomes in primary BC. In the present study, CTC EMT positivity (hazard ratio (HR) = 2.4; 95% CI 1.20-4.66, p = 0.013) and elevated neutrophil-to-lymphocyte ratio (NLR) (HR = 2.20; 95% CI 1.07-4.55; p = 0.033) were associated with shorter progression-free survival (PFS) in primary BC patients. Multivariate analysis showed that CTC EMT-positive patients with NLR ≥ 3 had 8.6 times increased risk of disease recurrence (95% CI 2.35-31.48, p = 0.001) compared with CTC EMT-negative patients with NLR < 3. Similarly, disease recurrence was 13.14 times more likely in CTC EMT-positive patients with MLR ≥ 0.34 (95% CI 4.35-39.67, p < 0.001). Given its low methodological and financial demands, the CBC-derived inflammation-based score determination could, after broader validation, significantly improve the prognostication of BC patients.

During cancer progression, breast tumor cells interact with adjacent adipose tissue, which has been shown to be engaged in cancer aggressiveness. However, the tumor-directed changes in adipose tissue-resident stromal cells affected by the tumor-stroma communication are still poorly understood. The acquired changes might remain in the tissue even after tumor removal and may contribute to tumor relapse. We investigated functional properties (migratory capacity, expression and secretion profile) of mesenchymal stromal cells isolated from healthy ( n = 9) and tumor-distant breast adipose tissue ( n = 32). Cancer patient-derived mesenchymal stromal cells (MSCs) (MSC-CA) exhibited a significantly disarranged secretion profile and proliferation potential. Co-culture with MDA-MB-231, T47D and JIMT-1, representing different subtypes of breast cancer, was used to analyze the effect of MSCs on proliferation, invasion and tumorigenicity. The MSC-CA enhanced tumorigenicity and altered xenograft composition in immunodeficient mice. Histological analysis revealed collective cell invasion with a specific invasive front of EMT-positive tumor cells as well as invasion of cancer cells to the nerve-surrounding space. This study identifies that adipose tissue-derived mesenchymal stromal cells are primed and permanently altered by tumor presence in breast tissue and have the potential to increase tumor cell invasive ability through the activation of epithelial-to-mesenchymal transition in tumor cells.

Microstructural refinement of magnesium (Mg) alloys is beneficial for mechanical and corrosion properties, both of which are critical for their successful application as temporary implant materials. One method of achieving a refined microstructure is through rapid solidification via gas-atomization-powder production. In this study we investigated spark plasma sintering (SPS) as a potential processing method for maintaining this refined microstructure while achieving a range of porosities up to full densification. We characterized the microstructural evolution as a function of sintering temperature from 250 to 450 °C for the alloy WE43 using multi-scale correlative microscopy techniques, including light microscopy and scanning and transmission electron microscopy-based methods. The spatial distribution of the two major alloying elements, neodymium (Nd) and yttrium (Y), was determined and the intermetallic phases they form identified using energy dispersive X-ray spectroscopy in conjunction with electron diffraction. The gas-atomized powder microstructure consists of Mg-rich dendrites and a percolating interdendritic Mg-Nd-Y ternary phase with structure Mg 14 Nd 2 Y, surrounded by a high Nd and Y content in solid solution. This microstructure is maintained up to a sintering temperature of 350 °C, while with higher sintering temperatures segregation of Nd and Y dominates. The percolating ternary phase breaks up into faceted globular precipitates with structure Mg 5 Nd, which is isomorphous to Mg 14 Nd 2 Y. Y comes out of solution and migrates to previous powder-particle surfaces, possibly forming Y 2 O 3 . Sample densities ranged from 64 to 100% for sintering temperatures of 250 to 450 °C, respectively, and the grain size remained constant at about 10 µm. SPS is demonstrated to be an attractive alternative method for processing Mg alloys to a wide range of porosities and fine microstructures. The microstructural refinement achieved by SPS holds the potential for slow and homogeneous corrosion. STATEMENT OF SIGNIFICANCE: This study presents the impact spark plasma sintering (SPS) has on the microstructure of WE43, a magnesium alloy used for biodegradable implants. SPS is of great interest in this context as it is scalable, rapid, and has the potential for tuning density while maintaining a refined microstructure. The microstructure and density are explored from the gas-atomized powder to the densified material using electron microscopy and chemical mapping from the macro- to the nano-level. The insights gained reveal an original evolution of rare-earth element distribution with an isomorphous chemistry change, while the microstructure develops from the non-equilibrium state (powder) towards an equilibrium structure upon sintering. This study, including measurements of mechanical performance, sets the premises of SPS for the fabrication of Mg-based implants with tunable characteristics., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Biodegradable magnesium alloys generally contain intermetallic phases on the micro- or nanoscale, which can initiate and control local corrosion processes via microgalvanic coupling. However, the experimental difficulties in characterizing active degradation on the nanoscale have so far limited the understanding of how these materials degrade in complex physiological environments. Here a quasi-in situ experiment based on transmission electron microscopy (TEM) is designed, which enables the initial corrosion attack at nanometric particles to be accessed within the first seconds of immersion. Combined with high-resolution ex situ cross-sectional TEM analysis of a well-developed corrosion-product layer, mechanistic insights into Mg-alloys' degradation on the nanoscale are provided over a large range of immersion times. Applying this methodology to lean Mg-Zn-Ca alloys and following in detail the dissolution of their nanometric Zn- and Ca-rich particles the in statu nascendi observation of intermetallic-particle dealloying is documented for magnesium alloys, where electrochemically active Ca and Mg preferentially dissolve and electropositive Zn enriches, inducing the particles' gradual ennoblement. Based on electrochemical theory, here, the concept of cathodic-polarization-induced dealloying, which controls the dynamic microstructural changes, is presented. The general prerequisites for this new dealloying mechanism to occur in multicomponent alloys and its distinction to other dealloying modes are also discussed., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Porosity is an essential feature in a wide range of applications that combine light weight with high surface area and tunable density. Porous materials can be easily prepared with a vast variety of chemistries using the salt-leaching technique. However, this templating approach has so far been limited to the fabrication of structures with random porosity and relatively simple macroscopic shapes. Here, a technique is reported that combines the ease of salt leaching with the complex shaping possibilities given by additive manufacturing (AM). By tuning the composition of surfactant and solvent, the salt-based paste is rheologically engineered and printed via direct ink writing into grid-like structures displaying structured pores that span from the sub-millimeter to the macroscopic scale. As a proof of concept, dried and sintered NaCl templates are infiltrated with magnesium (Mg), which is typically highly challenging to process by conventional AM techniques due to its highly oxidative nature and high vapor pressure. Mg scaffolds with well-controlled, ordered porosity are obtained after salt removal. The tunable mechanical properties and the potential to be predictably bioresorbed by the human body make these Mg scaffolds attractive for biomedical implants and demonstrate the great potential of this additive technique., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A Disintegrin And Metalloprotease 23 (ADAM23), a member of the ADAM family, is involved in neuronal differentiation and cancer. ADAM23 is considered a possible tumor suppressor gene and is frequently downregulated in various types of malignancies. Its epigenetic silencing through promoter hypermethylation was observed in breast cancer (BC). In the present study, we evaluated the prognostic significance of ADAM23 promoter methylation for hematogenous spread and disease-free survival (DFS). Pyrosequencing was used to quantify ADAM23 methylation in tumors of 203 BC patients. Presence of circulating tumor cells (CTC) in their peripheral blood was detected by quantitative RT-PCR. Expression of epithelial (KRT19) or mesenchymal (epithelial-mesenchymal transition [EMT]-inducing transcription factors TWIST1, SNAI1, SLUG and ZEB1) mRNA transcripts was examined in CD45-depleted peripheral blood mononuclear cells. ADAM23 methylation was significantly lower in tumors of patients with the mesenchymal CTC (P = .006). It positively correlated with Ki-67 proliferation, especially in mesenchymal CTC-negative patients (P = .001). In low-risk patients, characterized by low Ki-67 and mesenchymal CTC absence, ADAM23 hypermethylation was an independent predictor of DFS (P = .006). Our results indicate that ADAM23 is likely involved in BC progression and dissemination of mesenchymal CTC. ADAM23 methylation has the potential to function as a novel prognostic marker and therapeutic target., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Although solid tumors comprise malignant cells, they also contain many different non-malignant cell types in their micro-environment. The cellular components of the tumor stroma consist of immune and endothelial cells combined with a heterogeneous population of stromal cells which include cancer-associated fibroblasts. The bi-directional interactions between tumor and stromal cells therefore substantially affect tumor cell biology.Herein, we discuss current available information on these interactions in breast cancer chemo-resistance. It is acknowledged that stromal cells extrinsically alter tumor cell drug responses with profound consequences for therapy efficiency, and it is therefore essential to understand the molecular mechanisms which contribute to these substantial alterations because they provide potential targets for improved cancer therapy. Although breast cancer patient survival has improved over the last decades, chemo-resistance still remains a significant obstacle to successful treatment.Appreciating the important experimental evidence of mesenchymal stromal cells and cancer-associated fibroblast involvement in breast cancer clinical practice can therefore have important therapeutic implications.

Although the involvement of type 1 (IP 3 R1) and type 2 (IP 3 R2) inositol 1,4,5-trisphosphate receptors in apoptosis induction has been well documented in different cancer cells and tissues, the function of type 3 IP 3 R (IP 3 R3) is still elusive. Therefore, in this work we focused on the role of IP 3 R3 in tumor cells in vitro and in vivo. We determined increased expression of this receptor in clear cell renal cell carcinoma compared to matched unaffected part of the kidney from the same patient. Thus, we hypothesized about different functions of IP 3 R3 compared to IP 3 R1 and IP 3 R2 in tumor cells. Silencing of IP 3 R1 prevented apoptosis induction in colorectal cancer DLD1 cells, ovarian cancer A2780 cells, and clear cell renal cell carcinoma RCC4 cells, compared to apoptosis in cells treated with scrambled siRNA. As expected, silencing of IP 3 R3 and subsequent apoptosis induction resulted in increased levels of apoptosis in all these cells. Further, we prepared a DLD1/IP 3 R3&#95;del cell line using CRISPR/Cas9 gene editing method. These cells were injected into nude mice and tumor's volume was compared with tumors induced by DLD1 cells. Lower volume of tumors originated from DLD1/IP 3 R3&#95;del cells was observed after 12 days, compared to wild type DLD1 cells. Also, the migration of these cells was lesser compared to wild type DLD1 cells. Apoptosis under hypoxic conditions was more pronounced in DLD1/IP 3 R3&#95;del cells than in DLD1 cells. These results clearly show that IP 3 R3 has proliferative and anti-apoptotic effect in tumor cells, on contrary to the pro-apoptotic effect of IP 3 R1.

This paper studied the influence of two common sterilization techniques, ethylene oxide (EO) and gamma irradiation (GI), on the corrosion rate of four Mg-based materials in CO 2 -bicarbonate buffered Hanks' solution. The four materials were: high-purity (HP)-Mg, ZE41, ultra-high purity (XHP)-Mg, and XHP-ZX00. The corrosion rate was measured through mass loss (P m ) and hydrogen evolution (P H ). Two-way analysis of variance (ANOVA) was conducted to assess the effect of the sterilization techniques on the corrosion rates across the four materials. The ANOVA analyzed the variables of (1) material, (2) sterilization condition (EO, GI, and an unsterilized control group), and (3) the interaction between these two independent variables. Neither sterilization technique (EO and GI) significantly influenced the corrosion rate as measured by P m (p < 0.84) nor P H (p < 0.08). This result was consistent across the four materials tested, as there was no interaction between the test variables of material and sterilization condition for P m (p < 0.49) or P H (p < 0.27). As neither EO nor GI influenced the corrosion rates, either of these techniques warrants consideration for use on Mg-based medical implants and devices. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1907-1917, 2018., (© 2017 Wiley Periodicals, Inc.)

Mesenchymal stromal cells (MSCs) were introduced as tumor-targeted vehicles suitable for delivery of the gene-directed enzyme/prodrug therapy more than 10 years ago. Over these years key properties of tumor cells and MSCs, which are crucial for the treatment efficiency, were examined; and there are some critical issues to be considered for the maximum antitumor effect. Moreover, engineered MSCs expressing enzymes capable of activating non-toxic prodrugs achieved long-term curative effect even in metastatic and hard-to-treat tumor types in pre-clinical scenario(s). These gene-modified MSCs are termed prodrug-activating MSCs throughout the text and represent promising approach for further clinical application. This review summarizes major determinants to be considered for the application of the prodrug-activating MSCs in antitumor therapy in order to maximize therapeutic efficiency., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Porous titanium implants are widely used in dental, orthopaedic and otorhinolaryngology fields to improve implant integration to host tissue. A possible step further to improve the integration with the host is the incorporation of autologous cells in porous titanium structures via cell-laden hydrogels. Fast gelling hydrogels have advantageous properties for in situ applications such as localisation of specific cells and growth factors at a target area without dispersion. The ability to control the cell types in different regions of an implant is important in applications where the target tissue (i) has structural heterogeneity (multiple cell types with a defined spatial configuration with respect to each other); (ii) has physical property gradients essential for its function (such as in the case of osteochondral tissue transition). Due to their near immediate gelation, such gels can also be used for site-specific modification of porous titanium structures, particularly for implants which would face different tissues at different locations. Herein, we describe a step by step design of a model system: the model cell-laden gel-containing porous titanium implants in the form of titanium microbead/hydrogel (maleimide-dextran or maleimide-PVA based) microhybrids. These systems enable the determination of the effect of titanium presence on gel properties and encapsulated cell behaviour as a miniaturized version of full-scale implants, providing a system compatible with conventional analysis methods. We used a fibroblast/vascular endothelial cell co-cultures as our model system and by utilising single microbeads we have quantified the effect of gel microenvironment (degradability, presence of RGD peptides within gel formulation) on cell behaviour and the effect of the titanium presence on cell behaviour and gel formation. Titanium presence slightly changed gel properties without hindering gel formation or affecting cell viability. Cells showed a preference to move towards the titanium beads and fibroblast proliferation was significantly higher in hybrids compared to gel only controls. The MMP (Matrix Metalloproteinase)-sensitive hydrogels induced sprouting by cells in co-culture configuration which was quantified by fluorescence microscopy, confocal microscopy and qRT-PCR (Quantitative Reverse transcription polymerase chain reaction). When the microhybrid up-scaled to 3D thick structures, cellular localisation in specific areas of the 3D titanium structures was achieved, without decreasing overall cell proliferation compared to titanium only scaffolds. Microhybrids of titanium and hydrogels are useful models for deciding the necessary modifications of metallic implants and they can be used as a modelling system for the study of tissue/titanium implant interactions., Statement of Significance: This article demonstrates a method to apply cell-laden hydrogels to porous titanium implants and a model of titanium/hydrogel interaction at micro-level using titanium microbeads. The feasibility of site-specific modification of titanium implants with cell-laden microgels has been demonstrated. Use of titanium microbeads in combination with hydrogels with conventional analysis techniques as described in the article can facilitate the characterisation of surface modification of titanium in a relevant model system., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Suicide gene therapy mediated by mesenchymal stem cells with their ability to engraft into tumors makes these therapeutic stem cells an attractive tool to activate prodrugs directly within the tumor mass. In this study, we evaluated the therapeutic efficacy of human mesenchymal stem cells derived from bone marrow and from adipose tissue, engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase to treat intracerebral rat C6 glioblastoma in a simulated clinical therapeutic scenario. Intracerebrally grown glioblastoma was treated by resection and subsequently with single or repeated intracerebral inoculations of therapeutic stem cells followed by a continuous intracerebroventricular delivery of 5-fluorocytosine using an osmotic pump. Kaplan-Meier survival curves revealed that surgical resection of the tumor increased the survival time of the resected animals depending on the extent of surgical intervention. However, direct injections of therapeutic stem cells into the brain tissue surrounding the postoperative resection cavity led to a curative outcome in a significant number of treated animals. Moreover, the continuous supply of therapeutic stem cells into the brain with growing glioblastoma by osmotic pumps together with continuous prodrug delivery also proved to be therapeutically efficient. We assume that observed curative therapy of glioblastoma by stem cell-mediated prodrug gene therapy might be caused by the destruction of both tumor cells and the niche where glioblastoma initiating cells reside., (© 2013 UICC.)

Background: Application of autologous bone marrow mononuclear cells to "no option" patients with advanced critical limb ischemia (CLI) prevented major limb amputation in 73% patients during the 6-month follow-up. We examined which properties of bone marrow stromal cells also known as bone-marrow derived mesenchymal stem cells of responding and non-responding patients are important for amputation-free survival., Methods and Findings: Mesenchymal stem cells of 41 patients with CLI unsuitable for revascularisation were isolated from mononuclear bone marrow concentrate used for their treatment. Based on the clinical outcome of the treatment, we divided patients into two groups: responders and non-responders. Biological properties of responders' and non-responders' mesenchymal stem cells were characterized according to their ability to multiply, to differentiate in vitro, quantitative expression of cell surface markers, secretion of 27 cytokines, chemokines and growth factors, and to the relative expression of 15 mesenchymal stem cells important genes. Secretome comparison between responders (n=27) and non-responders (n=14) revealed significantly higher secretion values of IL-4, IL-6 and MIP-1b in the group of responders. The expression of cell markers CD44 and CD90 in mesenchymal stem cells from responders was significantly higher compared to non-responders (p<0.01). The expression of mesenchymal stem cells surface markers that was analyzed in 22 patients did not differ between diabetic (n=13) and non-diabetic (n=9) patient groups. Statistically significant higher expression of E-cadherin and PDX-1/IPF1 genes was found in non-responders, while expression of Snail was higher in responders., Conclusions: The quality of mesenchymal stem cells shown in the expression of cell surface markers, secreted factors and stem cell genes plays an important role in therapeutic outcome. Paracrine mechanisms are main drivers in the induction of reparatory processes in CLI patients. Differences in mesenchymal stem cells properties are discussed in relation to their involvement in the reparatory process.

Prodrug cancer gene therapy by mesenchymal stem cells (MSCs) targeted to tumors represents an attractive tool to activate prodrugs directly within the tumor mass, thus avoiding systemic toxicity. In this study, we tested the feasibility and efficacy of human adipose tissue-derived MSCs, engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase to treat intracranial rat C6 glioblastoma. Experiments were designed to simulate conditions of future clinical application for high-grade glioblastoma therapy by direct injections of therapeutic stem cells into tumor. We demonstrated that genetically modified therapeutic stem cells still have the tumor tropism when injected to a distant intracranial site and effectively inhibited glioblastoma growth after 5-fluorocytosine (5-FC) therapy. Coadministration of C6 cells and therapeutic stem cells with delayed 5-FC therapy improved the survival in a therapeutic stem cell dose-dependent manner and induced complete tumor regression in a significant number of animals. Continuous intracerebroventricular delivery of 5-FC using osmotic pump reduced the dose of prodrug required for the same therapeutic effect, and along with repeated administration of therapeutic stem cells increased the survival time. Intracerebral injection of therapeutic stem cells and treatment with 5-FC did not show any detectable adverse effects. Results support the arguments to begin clinical studies for treatment of high-grade brain tumors., (Copyright © 2011 UICC.)

The attractiveness of prodrug cancer gene therapy by stem cells targeted to tumors lies in activating the prodrug directly within the tumor mass, thus avoiding systemic toxicity. Suicide gene therapy using genetically engineered mesenchymal stem cells has the advantage of being safe, because prodrug administration not only eliminates tumor cells but consequently kills the more resistant therapeutic stem cells as well. This review provides an explanation of the stem cell-targeted prodrug cancer gene therapy principle, with focus on the choice of prodrug, properties of bone marrow and adipose tissue-derived mesenchymal stem and neural stem cells as well as the mechanisms of their tumor homing ability. Therapeutic achievements of the cytosine deaminase/5-fluorocytosine prodrug system and Herpes simplex virus thymidine kinase/ganciclovir are discussed. In addition, delivery of immunostimulatory cytokines, apoptosis inducing genes, nanoparticles and antiangiogenic proteins by stem cells to tumors and metastases is discussed as a promising approach for antitumor therapy. Combinations of traditional, targeted and stem cell-directed gene therapy could significantly advance the treatment of cancer.