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Abstract Background Pancreatic ductal adenocarcinoma (PDAC) poses a significant challenge due to late-stage diagnoses resulting from nonspecific early symptoms and the absence of early diagnostic biomarkers. MicroRNAs (miRNAs) play a crucial role in regulating diverse biological processes, and their abnormal expression is observed in various diseases, including cancer. Cancer stem cells (CSCs) are thought to act as a driving force in PDAC spread and recurrence. In pursuing the goal of unravelling the complexities of PDAC and its underlying molecular mechanisms, our study aimed to identify PDAC-associated miRNAs and relate them to disease progression, focusing on their involvement in various PDAC stages in patients and in reliable in vitro models, including pancreatic CSC (PaCSC) models. Methods The miRNA profiling datasets of serum and solid biopsies of PDAC patients deposited in GEO DataSets were analyzed by REML-based meta-analysis. The panel was then investigated by Real Time PCR in serum and solid biopsies of 37 PDAC patients enrolled in the study, as well as on BxPC-3 and AsPC-1 PDAC cell lines. We extended our focus towards a possible role of PDAC-associated miRNAs in the CSC phenotype, by inducing CSC-enriched pancreatospheres from BxPC-3 and AsPC-1 PDAC cell lines and performed differential miRNA expression analysis between PaCSCs and monolayer-grown PDAC cell lines. Results Meta-analysis showed differentially expressed miRNAs in blood samples and cancerous tissues of PDAC patients, allowing the identification of a panel of 9 PDAC-associated miRNAs. The results emerging from our patients fully confirmed the meta-analysis for the majority of miRNAs under investigation. In vitro tasks confirmed the aberrant expression of the panel of PDAC-associated miRNAs, with a dramatic dysregulation in PaCSC models. Notably, PaCSCs have shown significant overexpression of miR-4486, miR-216a-5p, and miR-216b-5p compared to PDAC cell lines, suggesting the recruitment of such miRNAs in stemness-related molecular mechanisms. Globally, our results showed a dual behaviour of miR-216a-5p and miR-216b-5p in PDAC while miR-4486, miR-361-3p, miR-125a-5p, miR-320d expression changes during the disease suggest they could promote PDAC initiation and progression. Conclusions This study contributed to an enhanced comprehension of the role of miRNAs in the development and progression of PDAC, shedding new light on the miRNA landscape in PDAC and its intricate interplay with CSCs, and providing specific insights useful in the development of miRNA-based diagnostic biomarkers and therapeutic targets.

Head and neck squamous cell carcinoma (HNSCC) presents a significant challenge worldwide due to its aggressiveness and high recurrence rates post-treatment, often linked to cancer stem cells (CSCs). Melatonin shows promise as a potent tumor suppressor; however, the effects of melatonin on CSCs remain unclear, and the development of models that closely resemble tumor heterogeneity could help to better understand the effects of this molecule. This study developed a tumor scaffold based on patient fibroblast-derived decellularized extracellular matrix that mimics the HNSCC microenvironment. Our study investigates the antitumoral effects of melatonin within this context. We validated its strong antiproliferative effect on HNSCC CSCs and the reduction of tumor invasion and migration markers, even in a strongly chemoprotective environment, as it is required to increase the minimum doses necessary to impact tumor viability compared to the non-scaffolded tumorspheres culture. Moreover, melatonin exhibited no cytotoxic effects on healthy cells co-cultured in the tumor hydrogel. This scaffold-based platform allows an in vitro study closer to HNSCC tumor reality, including CSCs, stromal component, and a biomimetic matrix, providing a new valuable research tool in precision oncology.

Abstract Macrophages infiltrating tumour tissues or residing in the microenvironment of solid tumours are known as tumour-associated macrophages (TAMs). These specialized immune cells play crucial roles in tumour growth, angiogenesis, immune regulation, metastasis, and chemoresistance. TAMs encompass various subpopulations, primarily classified into M1 and M2 subtypes based on their differentiation and activities. M1 macrophages, characterized by a pro-inflammatory phenotype, exert anti-tumoural effects, while M2 macrophages, with an anti-inflammatory phenotype, function as protumoural regulators. These highly versatile cells respond to stimuli from tumour cells and other constituents within the tumour microenvironment (TME), such as growth factors, cytokines, chemokines, and enzymes. These stimuli induce their polarization towards one phenotype or another, leading to complex interactions with TME components and influencing both pro-tumour and anti-tumour processes. This review comprehensively and deeply covers the literature on macrophages, their origin and function as well as the intricate interplay between macrophages and the TME, influencing the dual nature of TAMs in promoting both pro- and anti-tumour processes. Moreover, the review delves into the primary pathways implicated in macrophage polarization, examining the diverse stimuli that regulate this process. These stimuli play a crucial role in shaping the phenotype and functions of macrophages. In addition, the advantages and limitations of current macrophage based clinical interventions are reviewed, including enhancing TAM phagocytosis, inducing TAM exhaustion, inhibiting TAM recruitment, and polarizing TAMs towards an M1-like phenotype. In conclusion, while the treatment strategies targeting macrophages in precision medicine show promise, overcoming several obstacles is still necessary to achieve an accessible and efficient immunotherapy.

Hemophilia B is a genetic disorder characterized by clotting factor IX deficiency and bleeding in joints and muscles. Current treatments involve intravenous infusion of plasma-derived products or recombinant proteins, which have limited efficacy due to the short half-life of infused proteins. Recently, gene therapy for bleeding disorders has offered a potential solution. This study aimed to develop an in vitro gene therapy model using the CRISPR/Cas9 system to incorporate the F9 cDNA in human mesenchymal stem cells (hMSCs) to produce clotting factor IX. RNA guide sequences targeting the promoter-exon 1 region of the F9 gene were designed to incorporate a wild-type F9 cDNA into the cells. Knockin was performed with the CRISPR/Cas9 system and pDONOR-CMV/cDNAF9/IRES/EGFP vector template recombination in Lenti-X HEK293 cells and MSCs. A lentiviral F9 cDNA vector was designed as a FIX secretor model to validate the CRISPR/Cas9 system. Results showed successful gene editing and F9 expression in both cell models, although editing efficiency was lower in hMSCs. Future investigations will focus on improving gene editing efficiency using different transfection conditions or hybrid methodologies. This study demonstrates the potential of CRISPR/Cas9-based gene therapy in hMSCs as a target for hemophilia B. Further optimizations are required to translate these findings into clinical applications.

Background/Objectives: Three-dimensional bioprinting technology has enabled great advances in the treatment of articular cartilage (AC) defects by the biofabrication of biomimetic constructs that restore and/or regenerate damaged tissue. In this sense, the selection of suitable cells and biomaterials to bioprint constructs that mimic the architecture, composition, and functionality of the natural extracellular matrix (ECM) of the native tissue is crucial. In the present study, a novel cartilage-like biomimetic hybrid construct (CBC) was developed by 3D bioprinting to facilitate and promote AC regeneration. Methods: The CBC was biofabricated by the co-bioprinting of a bioink based on hyaluronic acid (HA) and alginate (AL) loaded with human mesenchymal stromal cells (hMSCs), with polylactic acid supporting the biomaterial, in order to mimic the microenvironment and structural properties of native AC, respectively. The CBC was biologically in vitro characterized. In addition, its physiochemical characteristics were evaluated in order to determine if the presence of hMSCs modified its properties. Results: Results from biological analysis demonstrated that CBC supported the high viability and proliferation of hMSCs, facilitating chondrogenesis after 5 weeks in vitro. The evaluation of physicochemical properties in the CBCs confirmed that the CBC developed could be suitable for use in cartilage tissue engineering. Conclusions: The results demonstrated that the use of bioprinted CBCs based on hMSC-AL/HA-bioink for AC repair could enhance the regeneration and/or formation of hyaline cartilaginous tissue.

Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of gene therapy approaches. Generally, inducible ON systems require a chimeric transcription factor (transactivator) that becomes activated by an inductor, which is not optimal for clinical translation due to their toxicity. We generated previously the first all-in-one, transactivator-free, doxycycline (Dox)-responsive (Lent-On-Plus or LOP) lentiviral vectors (LVs) able to control transgene expression in human stem cells. Here, we have generated new versions of the LOP LVs and have analyzed their applicability for the generation of inducible advanced therapy medicinal products (ATMPs) with special focus on primary human T cells. We have shown that, contrary to all other cell types analyzed, an Is2 insulator must be inserted into the 3′ long terminal repeat of the LOP LVs in order to control transgene expression in human primary T cells. Importantly, inducible primary T cells generated by the LOPIs2 LVs are responsive to ultralow doses of Dox and have no changes in phenotype or function compared with untransduced T cells. We validated the LOPIs2 system by generating inducible CAR-T cells that selectively kill CD19+ cells in the presence of Dox. In summary, we describe here the first transactivator-free, all-one-one system capable of generating Dox-inducible ATMPs.

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 7.5 mg/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.

The recent description of well-defined molecular subtypes of breast cancer has led to the clinical development of a number of successful molecular targets. Particularly, triple-negative breast cancer (TNBC) is an aggressive type of breast cancer with historically poor outcomes, mainly due to the lack of effective targeted therapies. Recent progresses in materials science have demonstrated the impressive properties of metal–organic framework nanoparticles (NPs) as antitumoral drug delivery systems. Here, in a way to achieve efficient bio-interfaces with cancer cells and improve their internalization, benchmarked MIL-100(Fe) NPs were coated with cell membranes (CMs) derived from the human TNBC cell line MDA-MB-468. The prepared CMs-coated metal–organic framework (CMs_MIL-100(Fe)) showed enhanced colloidal stability, cellular uptake, and cytotoxicity in MDA-MB-468 cells compared to non-coated NPs, paving the way for these human CMs-coated MIL-100(Fe) NPs as effective targeted therapies against the challenging TNBC.

To date, skin wounds are still an issue for healthcare professionals. Although numerous approaches have been developed over the years for skin regeneration, recent advances in regenerative medicine offer very promising strategies for the fabrication of artificial skin substitutes, including 3D bioprinting, electrospinning or spraying, among others. In particular, skin sprays are an innovative technique still under clinical evaluation that show great potential for the delivery of cells and hydrogels to treat acute and chronic wounds. Skin sprays present significant advantages compared to conventional treatments for wound healing, such as the facility of application, the possibility to treat large wound areas, or the homogeneous distribution of the sprayed material. In this article, we review the latest advances in this technology, giving a detailed description of investigational and currently commercially available acellular and cellular skin spray products, used for a variety of diseases and applying different experimental materials. Moreover, as skin sprays products are subjected to different classifications, we also explain the regulatory pathways for their commercialization and include the main clinical trials for different skin diseases and their treatment conditions. Finally, we argue and suggest possible future trends for the biotechnology of skin sprays for a better use in clinical dermatology.

Neoadjuvant chemotherapy (NACT) outcomes vary according to breast cancer (BC) subtype. Since pathologic complete response is one of the most important target endpoints of NACT, further investigation of NACT outcomes in BC is crucial. Thus, identifying sensitive and specific predictors of treatment response for each phenotype would enable early detection of chemoresistance and residual disease, decreasing exposures to ineffective therapies and enhancing overall survival rates. We used liquid chromatography−high‐resolution mass spectrometry (LC‐HRMS)‐based untargeted metabolomics to detect molecular changes in plasma of three different BC subtypes following the same NACT regimen, with the aim of searching for potential predictors of response. The metabolomics data set was analyzed by combining univariate and multivariate statistical strategies. By using ANOVA–simultaneous component analysis (ASCA), we were able to determine the prognostic value of potential biomarker candidates of response to NACT in the triple‐negative (TN) subtype. Higher concentrations of docosahexaenoic acid and secondary bile acids were found at basal and presurgery samples, respectively, in the responders group. In addition, the glycohyocholic and glycodeoxycholic acids were able to classify TN patients according to response to treatment and overall survival with an area under the curve model > 0.77. In relation to luminal B (LB) and HER2+ subjects, it should be noted that significant differences were related to time and individual factors. Specifically, tryptophan was identified to be decreased over time in HER2+ patients, whereas LysoPE (22:6) appeared to be increased, but could not be associated with response to NACT. Therefore, the combination of untargeted‐based metabolomics along with longitudinal statistical approaches may represent a very useful tool for the improvement of treatment and in administering a more personalized BC follow‐up in the clinical practice.

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. Most CAR-T cells 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 their moderate and TCR-like expression profile. Compared with CAR-T cells generated with human elongation factor α (EF1α)-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-α) 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 of AW-CAR-T cells in guanosine monophosphate (GMP)-like conditions. Based on these data, we propose the use of AW-LVs for the generation of improved CAR-T products.

Cardiovascular diseases (CVD) are the leading causes of death and disability in the world. Among all CVD, the most common is coronary artery disease (CAD). CAD results from the complications promoted by atherosclerosis, which is characterized by the accumulation of atherosclerotic plaques that limit and block the blood flow of the arteries involved in heart oxygenation. Atherosclerotic disease is usually treated by stents implantation and angioplasty, but these surgical interventions also favour thrombosis and restenosis which often lead to device failure. Hence, efficient and long-lasting therapeutic options that are easily accessible to patients are in high demand. Advanced technologies including nanotechnology or vascular tissue engineering may provide promising solutions for CVD. Moreover, advances in the understanding of the biological processes underlying atherosclerosis can lead to a significant improvement in the management of CVD and even to the development of novel efficient drugs. To note, over the last years, the observation that inflammation leads to atherosclerosis has gained interest providing a link between atheroma formation and oncogenesis. Here, we have focused on the description of the available therapy for atherosclerosis, including surgical treatment and experimental treatment, the mechanisms of atheroma formation, and possible novel therapeutic candidates such as the use of anti-inflammatory treatments to reduce CVD.

Abstract It has been well documented that the tumor microenvironment (TME) plays a key role in the promotion of drug resistance, the support of tumor progression, invasiveness, metastasis, and even the maintenance of a cancer stem-like phenotype. Here, we reviewed TME formation presenting it as a reflection of a tumor’s own organization during the different stages of tumor development. Interestingly, functionally different groups of stromal cells seem to have specific spatial distributions within the TME that change as the tumor evolves into advanced stage progression which correlates with the fact that cancer stem-like cells (CSCs) are located in the edges of solid tumor masses in advanced tumors. We also focus on the continuos feedback that is established between a tumor and its surroundings. The “talk” between tumor mass cells and TME stromal cells, marks the evolution of both interlocuting cell types. For instance, the metabolic and functional transformations that stromal cells undergo due to tumor corrupting activity. Moreover, the molecular basis of metastatic spread is also approached, making special emphasis on the site-specific pre-metastatic niche formation as another reflection of the primary tumor molecular signature. Finally, several therapeutic approaches targeting primary TME and pre-metastatic niche are suggested. For instance, a systematic analysis of the TME just adjacent to the tumor mass to establish the proportion of myofibroblasts-like cancer-associated fibroblasts (CAFs) which may in turn correspond to stemness and metastases-promotion. Or the implementation of “re-education” therapies consisting of switching tumor-supportive stromal cells into tumor-suppressive ones. In summary, to improve our clinical management of cancer, it is crucial to understand and learn how to manage the close interaction between TME and metastasis.

Abstract Cancer stem cells (CSCs) represent a tumor subpopulation responsible for tumor metastasis and resistance to chemo- and radiotherapy, ultimately leading to tumor relapse. As a consequence, the detection and eradication of this cell subpopulation represent a current challenge in oncology medicine. CSC phenotype is dependent on the tumor microenvironment (TME), which involves stem and differentiated tumor cells, as well as different cell types, such as mesenchymal stem cells, endothelial cells, fibroblasts and cells of the immune system, in addition to the extracellular matrix (ECM), different in composition to the ECM in healthy tissues. CSCs regulate multiple cancer hallmarks through the interaction with cells and ECM in their environment by secreting extracellular vesicles including exosomes, and soluble factors such as interleukins, cytokines, growth factors and other metabolites to the TME. Through these factors, CSCs generate and activate their own tumor niche by recruiting stromal cells and modulate angiogenesis, metastasis, resistance to antitumor treatments and their own maintenance by the secretion of different factors such as IL-6, VEGF and TGF-ß. Due to the strong influence of the CSC secretome on disease development, the new antitumor therapies focus on targeting these communication networks to eradicate the tumor and prevent metastasis, tumor relapse and drug resistance. This review summarizes for the first time the main components of the CSC secretome and how they mediate different tumor processes. Lastly, the relevance of the CSC secretome in the development of more precise and personalized antitumor therapies is discussed.

In breast cancer (BC), the presence of cancer stem cells (CSCs) has been related to relapse, metastasis, and radioresistance. Radiotherapy (RT) is an extended BC treatment, but is not always effective. CSCs have several mechanisms of radioresistance in place, and some miRNAs are involved in the cellular response to ionizing radiation (IR). Here, we studied how IR affects the expression of miRNAs related to stemness in different molecular BC subtypes. Exposition of BC cells to radiation doses of 2, 4, or 6 Gy affected their phenotype, functional characteristics, pluripotency gene expression, and in vivo tumorigenic capacity. This held true for various molecular subtypes of BC cells (classified by ER, PR and HER‐2 status), and for BC cells either plated in monolayer, or being in suspension as mammospheres. However, the effect of IR on the expression of eight stemness‐ and radioresistance‐related miRNAs (miR‐210, miR‐10b, miR‐182, miR‐142, miR‐221, miR‐21, miR‐93, miR‐15b) varied, depending on cell line subpopulation and clinicopathological features of BC patients. Therefore, clinicopathological features and, potentially also, chemotherapy regimen should be both taken into consideration, for determining a potential miRNA signature by liquid biopsy in BC patients treated with RT. Personalized and precision RT dosage regimes could improve the prognosis, treatment, and survival of BC 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 red-shifted 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.

Abstract Cancer stem cells (CSCs) subpopulation within the tumour is responsible for metastasis and cancer relapse. Here we investigate in vitro and in vivo the effects of a pancreatic (pro)enzyme mixture composed of Chymotrypsinogen and Trypsinogen (PRP) on CSCs derived from a human pancreatic cell line, BxPC3. Exposure of pancreatic CSCs spheres to PRP resulted in a significant decrease of ALDEFLUOR and specific pancreatic CSC markers (CD 326, CD 44 and CxCR4) signal tested by flow cytometry, further CSCs markers expression was also analyzed by western and immunofluorescence assays. PRP also inhibits primary and secondary sphere formation. Three RT2 Profiler PCR Arrays were used to study gene expression regulation after PRP treatment and resulted in, (i) epithelial-mesenchymal transition (EMT) inhibition; (ii) CSCs related genes suppression; (iii) enhanced expression of tumour suppressor genes; (iv) downregulation of migration and metastasis genes and (v) regulation of MAP Kinase Signalling Pathway. Finally, in vivo anti-tumor xenograft studies demonstrated high anti-tumour efficacy of PRP against tumours induced by BxPC3 human pancreatic CSCs. PRP impaired engrafting of pancreatic CSC’s tumours in nude mice and displayed an antigrowth effect toward initiated xenografts. We concluded that (pro)enzymes treatment is a valuable strategy to suppress the CSC population in solid pancreatic tumours.

There is a global need to discover effective anti-cancerous compounds from natural sources. Cultivated wheat cells can be a valuable source of non-toxic or low toxic plant-derived polysaccharides. In this study, we evaluated the anti-cancer ability of seven fractions of wheat cell culture polysaccharides (WCCPSs) in the HCT-116 colon cancer cell line. Almost all (6/7) fractions had an inhibitory effect on the proliferation of colon cancer cells, and two fractions (A-b and A-f) had considerable therapeutic indexes. The WCCPS fractions induced cell cycle arrest in the G1 phase and induced different rates of apoptosis (≤48%). Transmission and scanning electron microscopy revealed that WCCPS fractions caused apoptotic changes in the nucleus and cytoplasm, including damage to mitochondria and external morphological signs of apoptosis. In addition, the WCCPSs induced an increase in the levels of Bax, cytochrome c, and caspases 8 and 3, indicating that cell death progressed through intrinsic and extrinsic pathways of apoptosis. Furthermore, some fractions caused a significant decrease of c-Myc, b-catenin, NFkB2, and HCAM (CD 44) levels, indicating enhanced cell differentiation. Thus, for the first time, our results provide a proof of concept of the anti-cancer capacity of WCCPS fractions in colorectal cancer.

Abstract Tissue engineering (TE) seeks to fabricate implants that mimic the mechanical strength, structure, and composition of native tissues. Cartilage TE requires the development of functional personalized implants with cartilage‐like mechanical properties capable of sustaining high load‐bearing environments to integrate into the surrounding tissue of the cartilage defect. In this study, we evaluated the novel 1,4‐butanediol thermoplastic polyurethane elastomer (b‐TPUe) derivative filament as a 3D bioprinting material with application in cartilage TE. The mechanical behavior of b‐TPUe in terms of friction and elasticity were examined and compared with human articular cartilage, PCL, and PLA. Moreover, infrapatellar fat pad‐derived human mesenchymal stem cells (MSCs) were bioprinted together with scaffolds. in vitro cytotoxicity, proliferative potential, cell viability, and chondrogenic differentiation were analyzed by Alamar blue assay, SEM, confocal microscopy, and RT‐qPCR. Moreover, in vivo biocompatibility and host integration were analyzed. b‐TPUe demonstrated a much closer compression and shear behavior to native cartilage than PCL and PLA, as well as closer tribological properties to cartilage. Moreover, b‐TPUe bioprinted scaffolds were able to maintain proper proliferative potential, cell viability, and supported MSCs chondrogenesis. Finally, in vivo studies revealed no toxic effects 21 days after scaffolds implantation, extracellular matrix deposition and integration within the surrounding tissue. This is the first study that validates the biocompatibility of b‐TPUe for 3D bioprinting. Our findings indicate that this biomaterial can be exploited for the automated biofabrication of artificial tissues with tailorable mechanical properties including the great potential for cartilage TE applications.

Biofabrication technologies that use light for polymerization of biomaterials have made significant progress in the quality, resolution, and generation of precise complex tissue structures. In recent years, the evolution of these technologies has been growing along with the development of new photocurable resins and photoinitiators that are biocompatible and biodegradable with bioactive properties. Such evolution has allowed the progress of a large number of tissue engineering applications. Flexibility in the design, scale, and resolution and wide applicability of technologies are strongly dependent on the understanding of the biophysics involved in the biofabrication process. In particular, understanding cell–light interactions is crucial when bioprinting using cell-laden biomaterials. Here, we summarize some theoretical mechanisms, which condition cell response during bioprinting using light based technologies. We take a brief look at the light–biomaterial interaction for a better understanding of how linear effects (refraction, reflection, absorption, emission, and scattering) and nonlinear effects (two-photon absorption) influence the biofabricated tissue structures and identify the different parameters essential for maintaining cell viability during and after bioprinting.

Bell peppers are one of the most important species consumed and cultivated in Spain. Peppers are a source of carotenoids and phenolic compounds widely associated with biological activities such as antimicrobial, antiseptic, anticancer, counterirritant, cardioprotective, appetite stimulator, antioxidant, and immunomodulator. However, undersized and damaged fruits are usually wasted. Thus, in order to evaluate the phenolic content, a Box–Behnken design has been carried out to optimize the extraction from Capsicum annuum yellow pepper by ultrasound-assisted extraction (UAE). The independent factors were time (min), ethanol/water (% v/v) and solvent/sample ratio (v/w). The model was validated by ANOVA and confirmed. Furthermore, the whole pepper and the pepper without peduncles and seeds were extracted using optimal conditions and characterized by HPLC-ESI-TOF-MS. Moreover, their antioxidant activities, measured by three different methods (DPPH, ABTS, and FRAP), carotenoid composition, assessed by HPLC-MS, and chlorophyll content, assessed by a spectrophotometric method, were compared. A total of 38 polar compounds were found of which seven have been identified in pepper fruit extracts for the first time. According to the results, whole pepper (WP) samples presented higher content in phenolic acids; meanwhile, the edible portion (EP) was higher in flavonoids. No differences were found in the antioxidant activity except for the FRAP assay where the WP sample showed higher radical scavenging activity. EP samples showed the highest content of carotenoids and WP ones in chlorophylls.

Integration-deficient lentiviral vectors (IDLVs) have recently generated increasing interest, not only as a tool for transient gene delivery, but also as a technique for detecting off-target cleavage in gene-editing methodologies which rely on customized endonucleases (ENs). Despite their broad potential applications, the efficacy of IDLVs has historically been limited by low transgene expression and by the reduced sensitivity to detect low-frequency off-target events. We have previously reported that the incorporation of the chimeric sequence element IS2 into the long terminal repeat (LTR) of IDLVs increases gene expression levels, while also reducing the episome yield inside transduced cells. Our study demonstrates that the effectiveness of IDLVs relies on the balance between two parameters which can be modulated by the inclusion of IS2 sequences. In the present study, we explore new IDLV configurations harboring several elements based on IS2 modifications engineered to mediate more efficient transgene expression without affecting the targeted cell load. Of all the insulators and configurations analysed, the insertion of the IS2 into the 3′LTR produced the best results. After demonstrating a DAPI-low nuclear gene repositioning of IS2-containing episomes, we determined whether, in addition to a positive effect on transcription, the IS2 could improve the capture of IDLVs on double strand breaks (DSBs). Thus, DSBs were randomly generated, using the etoposide or locus-specific CRISPR-Cas9. Our results show that the IS2 element improved the efficacy of IDLV DSB detection. Altogether, our data indicate that the insertion of IS2 into the LTR of IDLVs improved, not only their transgene expression levels, but also their ability to be inserted into existing DSBs. This could have significant implications for the development of an unbiased detection tool for off-target cleavage sites from different specific nucleases.

Radiotherapy (RT) is a modality of oncologic treatment that can be used to treat approximately 50% of all cancer patients either alone or in combination with other treatment modalities such as surgery, chemotherapy, immunotherapy, and therapeutic targeting. Despite the technological advances in RT, which allow a more precise delivery of radiation while progressively minimizing the impact on normal tissues, issues like radioresistance and tumor recurrence remain important challenges. Tumor heterogeneity is responsible for the variation in the radiation response of the different tumor subpopulations. A main factor related to radioresistance is the presence of cancer stem cells (CSC) inside tumors, which are responsible for metastases, relapses, RT failure, and a poor prognosis in cancer patients. The plasticity of CSCs, a process highly dependent on the epithelial–mesenchymal transition (EMT) and associated to cell dedifferentiation, complicates the identification and eradication of CSCs and it might be involved in disease relapse and progression after irradiation. The tumor microenvironment and the interactions of CSCs with their niches also play an important role in the response to RT. This review provides a deep insight into the characteristics and radioresistance mechanisms of CSCs and into the role of CSCs and tumor microenvironment in both the primary tumor and metastasis in response to radiation, and the radiobiological principles related to the CSC response to RT. Finally, we summarize the major advances and clinical trials on the development of CSC-based therapies combined with RT to overcome radioresistance. A better understanding of the potential therapeutic targets for CSC radiosensitization will provide safer and more efficient combination strategies, which in turn will improve the live expectancy and curability of cancer patients.

The chemotherapeutic drug 5-FU is widely used in the treatment of a range of cancers, but resistance to the drug remains a major clinical problem. Since defects in the mediators of apoptosis may account for chemo-resistance, the identification of new targets involved in 5-FU-induced apoptosis is of main clinical interest. We have identified the ds-RNA-dependent protein kinase (PKR) as a key molecular target of 5-FU involved in apoptosis induction in human colon and breast cancer cell lines. PKR distribution and activation, apoptosis induction and cytotoxic effects were analyzed during 5-FU and 5-FU/IFNα treatment in several colon and breast cancer cell lines with different p53 status. PKR protein was activated by 5-FU treatment in a p53-independent manner, inducing phosphorylation of the protein synthesis translation initiation factor eIF-2α and cell death by apoptosis. Furthermore, PKR interference promoted a decreased response to 5-FU treatment and those cells were not affected by the synergistic antitumor activity of 5-FU/IFNα combination. These results, taken together, provide evidence that PKR is a key molecular target of 5-FU with potential relevance in the clinical use of this drug.

Colorectal cancer (CRC) is one of most common cancers worldwide, with high rates of mortality. Epidemiological findings demonstrate that coffee consumption reduces the risk of developing CRC by ~13%. In general, in vivo and in vitro findings demonstrate the antiproliferative, antioxidant and proapoptotic effects of brewed coffee or major bioavailable coffee compounds. Thus, it was assessed whether caffeine (CAF) and/or chlorogenic acid (CGA) attenuates the early-stage of chemically induced mouse colon carcinogenesis. Male Swiss mice were submitted to a 1,2-dimethylhydrazine/deoxycholic acid (DMH/DCA)-induced colon carcinogenesis model. These animals received CAF (50 mg/kg), CGA (25 mg/kg) or CAF+CGA (50 + 25 mg/kg) intragastrically for five times/week for ten weeks. CAF+CGA had the most pronounced effects on decreasing epithelial cell proliferation (Ki-67) and increasing apoptosis (cleaved caspase-3) in colonic crypts. This treatment also decreased the levels of proinflammatory cytokines IL-6, IL-17 and TNF-α, and downregulated the oncomiR miR-21a-5p in the colon. Accordingly, the analysis of miR-21a-5p targets demonstrated the genes involved in the negative regulation of proliferation and inflammation, and the positive regulation of apoptosis. Ultimately, CAF+CGA attenuated preneoplastic aberrant crypt foci (ACF) development. Our findings suggest that a combination of coffee compounds reduces early-stage colon carcinogenesis by the modulation of miR-21a-5p expression, highlighting the importance of coffee intake to prevent CRC. [ABSTRACT FROM AUTHOR]

• Human mesenchymal stem cells (MSCs) isolation methods affect the quality of their secretome. • MSCs isolated by the non-enzymatic methodology displayed higher cancer stem cells (CSCs) enrichment capacity. • MSCs isolated by the non-enzymatic methodology is more efficient in the CSCs growth maintenance, as show ALDH1+ levels. • The non-enzymatic methods is cheap and take less handling time. Malignant Melanoma is known for being one of the most aggressive cancers with an incidence that increases every year. Cancer stem cells (CSCs) are involved in the resistance to therapeutic treatments, the metastatic process, and the patient's relapses. Thus, it is of vital importance for researchers to find the methodology that allows us to obtain enriched subpopulations that maintain their stem-like properties without differentiating over time. In the present manuscript, our objective was to compare the ability of conditioned medium obtained from human mesenchymal stem cells (MSCs) isolated by enzymatic and non-enzymatic methods for the enrichment and maintenance of melanoma CSCs. Our results showed for the first time that MSCs isolated by less aggressive methodology displayed higher CSCs enrichment and maintenance capacity. Because they do not undergo enzymatic and proteolytic stress, MSCs produce a greater amount of signalling molecules, helping to improve the phenotype characteristics of CSCs and keep them over time. [ABSTRACT FROM AUTHOR]

In this study we evaluate the antitumour activity, the cell cycle arrest and apoptotic properties of novel lipophilic benzene-fused seven-membered 5-fluorouracil (5-FU) analogs in comparison to 5-FU on MCF-7 human breast cancer cells. The lipophilicities of ESB-786B, ESB-252A and ESB-928A were predicted by using the CDR option of the PALLAS 2.0 program. Cytotoxic assays were evaluated in MCF-7 cells treated with the sulforhodamine B colorimetric method. Cell cycle perturbations were studied by flow cytometry. Apoptosis was determined by both DNA fragmentation and annexin V-FITC and propidium iodide staining. The novel derivatives were more lipophilic than 5-FU and induced a marked growth inhibition, in a dose-dependent manner. After treatment with IC50 value (ranged from 2.5 to 22 μM) for each compound, light microscopy observation showed modifications in the morphology of MCF-7 cells. In addition, the 5-FU analogs arrest cells in the G0/G1 phase of the cell cycle whereas 5-FU induced arrest in S-phase. Moreover, induction of apoptosis was demonstrated by the annexin-V-based assay and confirmed using DNA fragmentation analysis on MCF-7 cells, a cell line in which the induction of DNA laddering is very difficult. The novel benzannelated seven-membered 5-FU analogs can be considered as specific apoptotic inducers. These experimental findings provide support for the use of these novel compounds as new weapons in the fight against breast cancer. [ABSTRACT FROM AUTHOR]