Glutathione metabolism is essential for self-renewal and chemoresistance of pancreatic cancer stem cells

BACKGROUND Cellular metabolism regulates stemness in health and disease. A reduced redox state is essential for self-renewal of normal and cancer stem cells (CSCs). However, while stem cells rely on glycolysis, different CSCs, including pancreatic CSCs, favor mitochondrial metabolism as their dominant energy-producing pathway. This suggests that powerful antioxidant networks must be in place to detoxify mitochondrial reactive oxygen species (ROS) and maintain stemness in oxidative CSCs. Since glutathione metabolism is critical for normal stem cell function and CSCs from breast, liver and gastric cancer show increased glutathione content, we hypothesized that pancreatic CSCs also rely on this pathway for ROS detoxification. AIM To investigate the role of glutathione metabolism in pancreatic CSCs. METHODS Primary pancreatic cancer cells of patient-derived xenografts (PDXs) were cultured in adherent or CSC-enriching sphere conditions to determine the role of glutathione metabolism in stemness. Real-time polymerase chain reaction (PCR) was used to validate RNAseq results involving glutathione metabolism genes in adherent vs spheres, as well as the expression of pluripotency-related genes following treatment. Public TCGA and GTEx RNAseq data from pancreatic cancer vs normal tissue samples were analyzed using the webserver GEPIA2. The glutathione-sensitive fluorescent probe monochlorobimane was used to determine glutathione content by fluorimetry or flow cytometry. Pharmacological inhibitors of glutathione synthesis and recycling [buthionine-sulfoximine (BSO) and 6-Aminonicotinamide (6-AN), respectively] were used to investigate the impact of glutathione depletion on CSC-enriched cultures. Staining with propidium iodide (cell cycle), Annexin-V (apoptosis) and CD133 (CSC content) were determined by flow cytometry. Self-renewal was assessed by sphere formation assay and response to gemcitabine treatment was used as a readout for chemoresistance. RESULTS Analysis of our previously published RNAseq dataset E-MTAB-3808 revealed up-regulation of genes involved in the KEGG (Kyoto Encyclopedia of Genes and Genomes) Pathway Glutathione Metabolism in CSC-enriched cultures compared to their differentiated counterparts. Consistently, in pancreatic cancer patient samples the expression of most of these up-regulated genes positively correlated with a stemness signature defined by NANOG, KLF4, SOX2 and OCT4 expression (P < 10-5). Moreover, 3 of the upregulated genes (MGST1, GPX8, GCCT) were associated with reduced disease-free survival in patients [Hazard ratio (HR) 2.2-2.5; P = 0.03-0.0054], suggesting a critical role for this pathway in pancreatic cancer progression. CSC-enriched sphere cultures also showed increased expression of different glutathione metabolism-related genes, as well as enhanced glutathione content in its reduced form (GSH). Glutathione depletion with BSO induced cell cycle arrest and apoptosis in spheres, and diminished the expression of stemness genes. Moreover, treatment with either BSO or the glutathione recycling inhibitor 6-AN inhibited self-renewal and the expression of the CSC marker CD133. GSH content in spheres positively correlated with intrinsic resistance to gemcitabine treatment in different PDXs r = 0.96, P = 5.8 × 1011). Additionally, CD133+ cells accumulated GSH in response to gemcitabine, which was abrogated by BSO treatment (P < 0.05). Combined treatment with BSO and gemcitabine-induced apoptosis in CD133+ cells to levels comparable to CD133- cells and significantly diminished self-renewal (P < 0.05), suggesting that chemoresistance of CSCs is partially dependent on GSH metabolism. CONCLUSION Our data suggest that pancreatic CSCs depend on glutathione metabolism. Pharmacological targeting of this pathway showed that high GSH content is essential to maintain CSC functionality in terms of self-renewal and chemoresistance

Descendants of the Jurassic turiasaurs from Iberia found refuge in the Early Cretaceous of western USA

A new, largely complete eusauropod dinosaur with cranial and postcranial elements from two skeletons, Mierasaurus bobyoungi gen. nov., sp. nov. from the lower Yellow Cat Member (Early Cretaceous) of Utah (USA), is the first recognized member of Turiasauria from North America. Moreover, according to our phylogenetic results, Moabosaurus utahensis from the lower Yellow Cat Member of Utah (USA) is also a member of this clade. This group of non-neosauropod eusauropods, which now includes five genera (Losillasaurus, Turiasaurus, Mierasaurus, Moabosaurus and Zby), was previously known only from the Jurassic of Europe. These recent discoveries in Utah suggest that turiasaurs as a lineage survived the Jurassic-Cretaceous extinction boundary and expanded their known range, at least, into western North America. The revised spatiotemporal distribution of turiasaurs is consistent with the presence of a land connection between North America and Europe sometime during the late Tithonian to Valanginian (c.147-133 Ma). Mierasaurus and Moabosaurus are the only non-neosauropod eusauropods known from North America, despite being younger than the classic neosauropods of the Morrison Formation (c.150 Ma)

A label free disposable device for rapid isolation of rare tumor cells from blood by ultrasounds

The use of blood samples as liquid biopsy is a label-free method for cancer diagnosis that offers benefits over traditional invasive biopsy techniques. Cell sorting by acoustic waves offers a means to separate rare cells from blood samples based on their physical properties in a label-free, contactless and biocompatible manner. Herein, we describe a flow-through separation approach that provides an efficient separation of tumor cells (TCs) from white blood cells (WBCs) in a microfluidic device, "THINUS-Chip" (Thin-Ultrasonic-Separator-Chip), actuated by ultrasounds. We introduce for the first time the concept of plate acoustic waves (PAW) applied to acoustophoresis as a new strategy. It lies in the geometrical chip design: different to other microseparators based on either bulk acoustic waves (BAW) or surface waves (SAW, SSAW and tSAW), it allows the use of polymeric materials without restrictions in the frequency of work. We demonstrate its ability to perform high-throughput isolation of TCs from WBCs, allowing a recovery rate of 84%±8% of TCs with a purity higher than 80% and combined viability of 85% at a flow rate of 80 µL/min (4.8 mL/h). The THINUS-Chip performs cell fractionation with low-cost manufacturing processes, opening the door to possible easy printing fabrication

Firms' Main Market, Human Capital and Wages

Recent international trade literature emphasizes two features in characterizing the current patterns of trade: efficiency heterogeneity at the firm level and quality differentiation. This paper explores human capital and wage differences across firms in that context. We build a partial equilibrium model predicting that firms selling in more-remote markets employ higher human capital and pay higher wages to employees within each education group. The channel linking these variables is firms’ endogenous choice of quality. Predictions are tested using Spanish employer-employee matched data that classify firms according to four main destination markets: local, national, European Union, and rest of the World. Employees’ average education is increasing in the remoteness of firm’s main output market. Market–destination wage premia are large, increasing in the remoteness of the market, and increasing in individual education. These results suggest that increasing globalization may play a significant role in raising wage inequality within and across education groups

An Aptamer against MNK1 for Non-Small Cell Lung Cancer Treatment

Lung cancer is the leading cause of cancer-related death worldwide. Its late diagnosis and consequently poor survival make necessary the search for new therapeutic targets. The mitogen-activated protein kinase (MAPK)-interacting kinase 1 (MNK1) is overexpressed in lung cancer and correlates with poor overall survival in non-small cell lung cancer (NSCLC) patients. The previously identified and optimized aptamer from our laboratory against MNK1, apMNKQ2, showed promising results as an antitumor drug in breast cancer in vitro and in vivo. Thus, the present study shows the antitumor potential of apMNKQ2 in another type of cancer where MNK1 plays a significant role, such as NSCLC. The effect of apMNKQ2 in lung cancer was studied with viability, toxicity, clonogenic, migration, invasion, and in vivo efficacy assays. Our results show that apMNKQ2 arrests the cell cycle and reduces viability, colony formation, migration, invasion, and epithelial-mesenchymal transition (EMT) processes in NSCLC cells. In addition, apMNKQ2 reduces tumor growth in an A549-cell line NSCLC xenograft model. In summary, targeting MNK1 with a specific aptamer may provide an innovative strategy for lung cancer treatment.R.C.-M. was supported for predoctoral contracts (PEJD 2016-BMD-2145 and 2018-BMD-9201) from the Community of Madrid and grant RTC2019-07227-1. M.E.M. and V.M.G. are researchers from FIBio-HRC supported by Consejeria de Sanidad (CAM). This work was supported by grants RTC2019-07227-1 and PID2019-105417RB-I00, funded by MCIN/AEI/10.13039/501100011033 (Ministry of Economy and Competitiveness, Spain)

ISG15 and ISGylation is required for pancreatic cancer stem cell mitophagy and metabolic plasticity

Pancreatic cancer stem cells (PaCSCs) drive pancreatic cancer tumorigenesis, chemoresistance and metastasis. While eliminating this subpopulation of cells would theoretically result in tumor eradication, PaCSCs are extremely plastic and can successfully adapt to targeted therapies. In this study, we demonstrate that PaCSCs increase expression of interferon-stimulated gene 15 (ISG15) and protein ISGylation, which are essential for maintaining their metabolic plasticity. CRISPR-mediated ISG15 genomic editing reduces overall ISGylation, impairing PaCSCs self-renewal and their in vivo tumorigenic capacity. At the molecular level, ISG15 loss results in decreased mitochondrial ISGylation concomitant with increased accumulation of dysfunctional mitochondria, reduced oxidative phosphorylation (OXPHOS) and impaired mitophagy. Importantly, disruption in mitochondrial metabolism affects PaCSC metabolic plasticity, making them susceptible to prolonged inhibition with metformin in vivo. Thus, ISGylation is critical for optimal and efficient OXPHOS by ensuring the recycling of dysfunctional mitochondria, and when absent, a dysregulation in mitophagy occurs that negatively impacts PaCSC stemness

Exploiting oxidative phosphorylation to promote the stem and immunoevasive properties of pancreatic cancer stem cells

Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer death, has a 5-year survival rate of approximately 7–9%. The ineffectiveness of anti-PDAC therapies is believed to be due to the existence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are functionally plastic, and have exclusive tumorigenic, chemoresistant and metastatic capacities. Herein, we describe a 2D in vitro system for long-term enrichment of pancreatic CSCs that is amenable to biological and CSC-specific studies. By changing the carbon source from glucose to galactose in vitro, we force PDAC cells to utilize OXPHOS, resulting in enrichment of CSCs defined by increased CSC biomarker and pluripotency gene expression, greater tumorigenic potential, induced but reversible quiescence, increased OXPHOS activity, enhanced invasiveness, and upregulated immune evasion properties. This CSC enrichment method can facilitate the discovery of new CSC-specific hallmarks for future development into targets for PDAC-based therapies

Exploiting oxidative phosphorylation to promote the stem and immunoevasive properties of pancreatic cancer stem cells

© The Author(s) 2020Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer death, has a 5-year survival rate of approximately 7–9%. The ineffectiveness of anti-PDAC therapies is believed to be due to the existence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are functionally plastic, and have exclusive tumorigenic, chemoresistant and metastatic capacities. Herein, we describe a 2D in vitro system for long-term enrichment of pancreatic CSCs that is amenable to biological and CSC-specific studies. By changing the carbon source from glucose to galactose in vitro, we force PDAC cells to utilize OXPHOS, resulting in enrichment of CSCs defined by increased CSC biomarker and pluripotency gene expression, greater tumorigenic potential, induced but reversible quiescence, increased OXPHOS activity, enhanced invasiveness, and upregulated immune evasion properties. This CSC enrichment method can facilitate the discovery of new CSC-specific hallmarks for future development into targets for PDAC-based therapies.We acknowledge and thank Dr. Nuria Malats and Jaime Villarreal from the Spanish National Cancer Research Center (CNIO) for RNA sequencing and analysis, funded by Fondo de Investigaciones Sanitarias (FIS) grant PI18/01347. We thank Patricia Sánchez-Tomero and Marina Ochando-Garmendia for technical assistance and support and Dr. Raúl Sánchez Lanzas for assistance with autophagy experiments. We want to particularly acknowledge the patients and the BioBank Hospital Ramón y Cajal-IRYCIS (PT13/0010/0002) integrated in the Spanish National Biobanks Network for its collaboration and, in particular, Adrián Povo Retana for macrophage isolation. We would also like to thank the Transmission Electron Microscopy Unit Laboratory, part of the UAM Interdepartmental Investigation Service (SIdI); Coral Pedrero for exceptional help with in vivo experiments; and the laboratories of Dr. Amparo Cano and Dr. José González Castaño for reagents and helpful discussions. S.V. was a recipient of an Ayuda de Movilidad del Personal Investigador del IRYCIS, a mobility grant from the Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain, and a pre-doctoral fellowship from the Comunidad de Madrid, Ayudas Para La Contratación De Investigadores Predoctorales Y Posdoctorales (PEJD-2017-PRE/BMD-5062), Madrid, Spain. This study was supported by a Rámon y Cajal Merit Award (RYC-2012-12104) from the Ministerio de Economía y Competitividad, Spain (to B.S.); funding from la Beca Carmen Delgado/Miguel Pérez-Mateo from AESPANC-ACANPAN Spain (to B.S.); a Conquer Cancer Now Grant from the Concern Foundation (Los Angeles, CA, USA) (to B.S.); a Coordinated grant (GC16173694BARB) from the Fundación Asociación Española Contra el Cáncer (AECC) (to B.S.); FIS grants PI18/00757 (to B.S.), PI16/00789 (to M.A.F.-M.), PI18/00267 (to L.G.-B.; co-financed through Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa”); a Miguel Servet award (CP16/00121) (to P.S.); a Max Eder Fellowship of the German Cancer Aid (111746) (to P.C.H.); and the German Research Foundation (DFG, CRC 1279 “Exploiting the human peptidome for Novel Antimicrobial and Anticancer Agents”; to P.C.H.)

DEDICATE: proposal for a conceptual framework to develop dementia-friendly Integrated eCare support

Background: Evidence shows that the implementation of Information and Communication Technologies (ICT) enabled services supporting integrated dementia care represents an opportunity that faces multi-pronged challenges. First, the provision of dementia support is fragmented and often inappropriate. Second, available ICT solutions in this field do not address the full spectrum of support needs arising across an individual’s whole dementia journey. Current solutions fail to harness the potential of available validated e-health services, such as telehealth and telecare, for the purposes of dementia care. Third, there is a lack of understanding of how viable business models in this field can operate. The field comprises both professional and non-professional players that interact and have roles to play in ensuring that useful technologies are developed, implemented and used. Methods: Starting from a literature review, including relevant pilot projects for ICT-based dementia care, we define the major requirements of a system able to overcome the limitations evidenced in the literature, and how this system should be integrated in the socio-technical ecosystem characterizing this disease. From here, we define the DEDICATE architecture of such a system, and the conceptual framework mapping the architecture over the requirements. Results: We identified three macro-requirements, namely the need to overcome: deficient technology innovation, deficient service process innovation, and deficient business models innovation. The proposed architecture is a three level architecture in which the center (data layer) includes patients’ and informal caregivers’ preferences, memories, and other personal data relevant to sustain the dementia journey, is connected through a middleware (service layer), which guarantees core IT services and integration, to dedicated applications (application layer) to sustain dementia care (Formal Support Services, FSS), and to existing formal care infrastructures, in order to guarantee care coordination (Care Coordination Services, CCS). Conclusions: The proposed DEDICATE architecture and framework envisages a feasible means to overcome the present barriers by: (1) developing and integrating technologies that can follow the patient and the caregivers throughout the development of the condition, since the early stages in which the patient is able to build up preferences and memories will be used in the later stages to maximise personalization and thereby improve efficacy and usability (technology innovation); (2) guaranteeing the care coordination between formal and informal caregivers, and giving an active yet supported role to the latter (service innovation); and (3) integrating existing infrastructures and care models to decrease the cost of the overall care pathway, by improving system interoperability (business model innovation)

Platinum iodido drugs show potential anti-tumor activity, affecting cancer cell metabolism and inducing ROS and senescence in gastrointestinal cancer cells

Cisplatin-based chemotherapy has associated clinical disadvantages, such as high toxicity and resistance. Thus, the development of new antitumor metallodrugs able to overcome different clinical barriers is a public healthcare priority. Here, we studied the mechanism of action of the isomers trans and cis-[PtI2(isopropylamine)2] (I5 and I6, respectively) against gastrointestinal cancer cells. We demonstrate that I5 and I6 modulate mitochondrial metabolism, decreasing OXPHOS activity and negatively affecting ATP-linked oxygen consumption rate. Consequently, I5 and I6 generated Reactive Oxygen Species (ROS), provoking oxidative damage and eventually the induction of senescence. Thus, herein we propose a loop with three interconnected processes modulated by these iodido agents: (i) mitochondrial dysfunction and metabolic disruptions; (ii) ROS generation and oxidative damage; and (iii) cellular senescence. Functionally, I5 reduces cancer cell clonogenicity and tumor growth in a pancreatic xenograft model without systemic toxicity, highlighting a potential anticancer complex that warrants additional pre-clinical studies.This research was funded by PID2019-106220RB100, RTC2019-007227-1 granted by MICIU/ AEI / 10.13039/501100011033, and PID2022-137373OB-I00 granted by MICIU/ AEI / 10.13039/501100011033 / FEDER, UE. This study was supported by ISCIII FIS grants PI20/0335 (RP) and PI21/01110 (BSJ), co-financed through Fondo Europeo de Desarrollo Regional (FEDER) ’Una manera de hacer Europa’. JMH was supported by a grant FPI-UAM 2021 from Universidad Autónoma de Madrid (Molecular BioSciences PhD program). We thank Gonzalo Vílchez and Sofia Figueiras for their contribution to this work via Comunidad de Madrid grants PEJ-2020-AI/BMD-18538; PEJ-2021-TL/BMD-22441.Peer reviewe