Physico-chemical characterization of the tumour microenvironment of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal malignancy that accounts for more than 90% of pancreatic cancer diagnoses. Our research is focused on the physico-chemical properties of the tumour microenvironment (TME), including its tumoural extracellular matrix (tECM), as they may have an important impact on the success of cancer therapies. PDAC xenografts and their decellularized tECM offer a great material source for research in terms of biomimicry with the original human tumour. Our aim was to evaluate and quantify the physico-chemical properties of the PDAC TME. Both cellularized (native TME) and decellularized (tECM) patient-derived PDAC xenografts were analyzed. A factorial design of experiments identified an optimal combination of factors for effective xenograft decellularization. Our results provide a complete advance in our understanding of the PDAC TME and its corresponding stroma, showing that it presents an interconnected porous architecture with very low permeability and small pores due to the contractility of the cellular components. This fact provides a potential therapeutic strategy based on the therapeutic agent size.

Inhibiting NR5A2 targets stemness in pancreatic cancer by disrupting SOX2/MYC signaling and restoring chemosensitivity.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a profoundly aggressive and fatal cancer. One of the key factors defining its aggressiveness and resilience against chemotherapy is the existence of cancer stem cells (CSCs). The important task of discovering upstream regulators of stemness that are amenable for targeting in PDAC is essential for the advancement of more potent therapeutic approaches. In this study, we sought to elucidate the function of the nuclear receptor subfamily 5, group A, member 2 (NR5A2) in the context of pancreatic CSCs. METHODS: We modeled human PDAC using primary PDAC cells and CSC-enriched sphere cultures. NR5A2 was genetically silenced or inhibited with Cpd3. Assays included RNA-seq, sphere/colony formation, cell viability/toxicity, real-time PCR, western blot, immunofluorescence, ChIP, CUT&Tag, XF Analysis, lactate production, and in vivo tumorigenicity assays. PDAC models from 18 patients were treated with Cpd3-loaded nanocarriers. RESULTS: Our findings demonstrate that NR5A2 plays a dual role in PDAC. In differentiated cancer cells, NR5A2 promotes cell proliferation by inhibiting CDKN1A. On the other hand, in the CSC population, NR5A2 enhances stemness by upregulating SOX2 through direct binding to its promotor/enhancer region. Additionally, NR5A2 suppresses MYC, leading to the activation of the mitochondrial biogenesis factor PPARGC1A and a shift in metabolism towards oxidative phosphorylation, which is a crucial feature of stemness in PDAC. Importantly, our study shows that the specific NR5A2 inhibitor, Cpd3, sensitizes a significant fraction of PDAC models derived from 18 patients to standard chemotherapy. This treatment approach results in durable remissions and long-term survival. Furthermore, we demonstrate that the expression levels of NR5A2/SOX2 can predict the response to treatment. CONCLUSIONS: The findings of our study highlight the cell context-dependent effects of NR5A2 in PDAC. We have identified a novel pharmacological strategy to modulate SOX2 and MYC levels, which disrupts stemness and prevents relapse in this deadly disease. These insights provide valuable information for the development of targeted therapies for PDAC, offering new hope for improved patient outcomes. A Schematic illustration of the role of NR5A2 in cancer stem cells versus differentiated cancer cells, along with the action of the NR5A2 inhibitor Cpd3. B Overall survival of tumor-bearing mice following allocated treatment. A total of 18 PDX models were treated using a 2 x 1 x 1 approach (two animals per model per treatment); n=36 per group (illustration created with biorender.com ).

Structural Validity and Internal Consistency of the Adolescents and Children Risk of Abuse and Maltreatment Parental Scale (ACRAM-PS).

There is a clear need for developing a comprehensive, unbiased, and psychometrically sound tool to assess child maltreatment. The aim of this study is to examine the structural validity, internal consistency, and convergent validity of a newly developed child maltreatment assessment instrument. A total of 286 professionals of the child protection system participated in the study, completing a total of 645 cases of children and adolescents. The Adolescents and Children Risk of Abuse and Maltreatment Parental Scale (ACRAM-PS), the Childhood Trauma Questionnaire Short Form (CTQ-SF) and other demographic variables were measured. Structural validity, internal consistency, and convergent validity of the ACRAM-PS were tested. This scale obtained good structural validity, internal consistency, and convergent validity as hypothesized patterns of correlations occurred as expected. This instrument implies a considerable improvement as it is comprehensive, psychometrically sound and, it has been articulated by its own users. It can significantly contribute to establish a common language among professionals, improve multidisciplinary communication, and optimize prevention, detection, and early intervention in child maltreatment.

Macrophages direct cancer cells through a LOXL2-mediated metastatic cascade in pancreatic ductal adenocarcinoma.

OBJECTIVE: The lysyl oxidase-like protein 2 (LOXL2) contributes to tumour progression and metastasis in different tumour entities, but its role in pancreatic ductal adenocarcinoma (PDAC) has not been evaluated in immunocompetent in vivo PDAC models. DESIGN: Towards this end, we used PDAC patient data sets, patient-derived xenograft in vivo and in vitro models, and four conditional genetically-engineered mouse models (GEMMS) to dissect the role of LOXL2 in PDAC. For GEMM-based studies, K-Ras +/LSL-G12D;Trp53 LSL-R172H;Pdx1-Cre mice (KPC) and the K-Ras +/LSL-G12D;Pdx1-Cre mice (KC) were crossed with Loxl2 allele floxed mice (Loxl2Exon2 fl/fl) or conditional Loxl2 overexpressing mice (R26Loxl2 KI/KI) to generate KPCL2KO or KCL2KO and KPCL2KI or KCL2KI mice, which were used to study overall survival; tumour incidence, burden and differentiation; metastases; epithelial to mesenchymal transition (EMT); stemness and extracellular collagen matrix (ECM) organisation. RESULTS: Using these PDAC mouse models, we show that while Loxl2 ablation had little effect on primary tumour development and growth, its loss significantly decreased metastasis and increased overall survival. We attribute this effect to non-cell autonomous factors, primarily ECM remodelling. Loxl2 overexpression, on the other hand, promoted primary and metastatic tumour growth and decreased overall survival, which could be linked to increased EMT and stemness. We also identified tumour-associated macrophage-secreted oncostatin M (OSM) as an inducer of LOXL2 expression, and show that targeting macrophages in vivo affects Osm and Loxl2 expression and collagen fibre alignment. CONCLUSION: Taken together, our findings establish novel pathophysiological roles and functions for LOXL2 in PDAC, which could be potentially exploited to treat metastatic disease.

Pharmacological targeting of the receptor ALK inhibits tumorigenicity and overcomes chemoresistance in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive disease characterized by its metastatic potential and chemoresistance. These traits are partially attributable to the highly tumorigenic pancreatic cancer stem cells (PaCSCs). Interestingly, these cells show unique features in order to sustain their identity and functionality, some of them amenable for therapeutic intervention. Screening of phospho-receptor tyrosine kinases revealed that PaCSCs harbored increased activation of anaplastic lymphoma kinase (ALK). We subsequently demonstrated that oncogenic ALK signaling contributes to tumorigenicity in PDAC patient-derived xenografts (PDXs) by promoting stemness through ligand-dependent activation. Indeed, the ALK ligands midkine (MDK) or pleiotrophin (PTN) increased self-renewal, clonogenicity and CSC frequency in several in vitro local and metastatic PDX models. Conversely, treatment with the clinically-approved ALK inhibitors Crizotinib and Ensartinib decreased PaCSC content and functionality in vitro and in vivo, by inducing cell death. Strikingly, ALK inhibitors sensitized chemoresistant PaCSCs to Gemcitabine, as the most used chemotherapeutic agent for PDAC treatment. Consequently, ALK inhibition delayed tumor relapse after chemotherapy in vivo by effectively decreasing the content of PaCSCs. In summary, our results demonstrate that targeting the MDK/PTN-ALK axis with clinically-approved inhibitors impairs in vivo tumorigenicity and chemoresistance in PDAC suggesting a new treatment approach to improve the long-term survival of PDAC patients.

El impacto de la soledad y la fragilidad en el bienestar de los mayores europeos / The impact of loneliness and frailty on the well-being of older Europeans

Resumen Introducción: El bienestar es un constructo importante cuyos determinantes asociados deben ser identificados para comprender con mayor profundidad los procesos para envejecer de forma saludable. El estudio del impacto de la fragilidad en el bienestar de las personas mayores requiere incorporar los dominios físico, psicológico y social. Así, el adulto mayor frágil puede definirse a partir de la fragilidad física, junto con la soledad como fragilidad social. Este estudio propone un modelo para estudiar el impacto de la fragilidad física y la soledad sobre el bienestar de las personas mayores, diferenciando entre control, autonomía, placer y autorrealización. Método: Se realizó un estudio de carácter transversal con una muestra de 13569 personas mayores provenientes de la encuesta SHARE, para estimar un modelo de ecuaciones estructurales en el que se contemplaban los efectos de la fragilidad y la soledad en el bienestar (cuestionario CASP), controlado por el género, la edad y la salud percibida. Resultados: El modelo final contemplaba el efecto del género sobre el indicador de fuerza de prensión de fragilidad. Los resultados mostraron un ajuste adecuado del modelo a los datos y consiguieron explicar un 80.1% de la varianza de control, un 48.6% de la de autonomía, un 43.6% de la de placer y un 61.3% de la varianza de autorrealización. Conclusiones: La soledad y la fragilidad presentaron efectos diferenciales en función del componente de bienestar. Se discuten las implicaciones de dichos efectos en el desarrollo de intervenciones.

Abstract Introduction: Well-being is an important construct whose associated determinants must be identified in order to further understand the processes for healthy aging. The study of the impact of frailty on older people's wellbeing requires the incorporation of physical, psychological and social domains. Hence, frail older adults can be defined from physical frailty, together with loneliness as social frailty. This study proposes a model to study the impact of frailty and loneliness on well-being of older adults, differentiating among control, autonomy, pleasure and self-realization. Method: A cross-sectional study was carried out with a sample of 13569 older adults from the SHARE project was employed in order to estimate a structural equation model in which frailty and loneliness affected well-being (CASP questionnaire), while controlling for gender, age and perceived health. Results: The final model additionally contemplated the effect of gender on the grip strength frailty indicator. Results showed a good fit of the model to the data and could explain 80.1% of the variance of control, 48.6% of autonomy's, 43.6% of pleasure's and 61.3% of the variance of self-realization. Conclusions: Loneliness and frailty showed differential effects on the different components of well-being. Implications of these effects on intervention development are discussed

Metabolic determinants of stemness in medulloblastoma.

Medulloblastomas (MBs) are the most prevalent brain tumours in children. They are classified as grade IV, the highest in malignancy, with about 30% metastatic tumours at the time of diagnosis. Cancer stem cells (CSCs) are a small subset of tumour cells that can initiate and support tumour growth. In MB, CSCs contribute to tumour initiation, metastasis, and therapy resistance. Metabolic differences among the different MB groups have started to emerge. Sonic hedgehog tumours show enriched lipid and nucleic acid metabolism pathways, whereas Group 3 MBs upregulate glycolysis, gluconeogenesis, glutamine anabolism, and glut athione-mediated anti-oxidant pathways. Such differences impact the clinical behaviour of MB tumours and can be exploited therapeutically. In this review, we summarise the existing knowledge about metabolic rewiring in MB, with a particular focus on MB-CSCs. Finally, we highlight some of the emerging metabolism-based therapeutic strategies for MB.

Measurement Invariance of the Brief Resilient Coping Scale (BRCS) in Peruvian and Spanish Older Adults.

Although the Brief Resilient Coping Scale (BRCS) has been validated in some European and American countries, there are no studies that evaluate its factorial invariance among different nations. In this sense, the objective of the study is to evaluate the factorial invariance of the BRCS in samples of older adults in Peru and Spain, using multigroup Confirmatory Factor Analysis. 236 older adults from Peru participated (Mean age = 72.8, SD = 6.90) and 133 older adults from Spain (Mean age = 71, SD = 7). In the Peruvian sample 78.4% were women and 21.6% men; while in the Spanish sample the majority were women (69.9%). The BRCS was scalar invariant but not strictly invariant between Spain and Peru. Our results found invariance of the structure, factor loadings and intercepts in both countries. These results support the use of BRCS in studies that compare the resilience between samples of older adults in both countries, and encourage applied research for the development of resilience in older adults in Spain and Peru.

Lipid droplets as metabolic determinants for stemness and chemoresistance in cancer.

Previously regarded as simple fat storage particles, new evidence suggests that lipid droplets (LDs) are dynamic and functional organelles involved in key cellular processes such as membrane biosynthesis, lipid metabolism, cell signalling and inflammation. Indeed, an increased LD content is one of the most apparent features resulting from lipid metabolism reprogramming necessary to support the basic functions of cancer cells. LDs have been associated to different cellular processes involved in cancer progression and aggressiveness, such as tumorigenicity, invasion and metastasis, as well as chemoresistance. Interestingly, all of these processes are controlled by a subpopulation of highly aggressive tumoral cells named cancer stem cells (CSCs), suggesting that LDs may be fundamental elements for stemness in cancer. Considering the key role of CSCs on chemoresistance and disease relapse, main factors of therapy failure, the design of novel therapeutic approaches targeting these cells may be the only chance for long-term survival in cancer patients. In this sense, their biology and functional properties render LDs excellent candidates for target discovery and design of combined therapeutic strategies. In this review, we summarise the current knowledge identifying LDs and CSCs as main contributors to cancer aggressiveness, metastasis and chemoresistance.

Glucose and Amino Acid Metabolic Dependencies Linked to Stemness and Metastasis in Different Aggressive Cancer Types.

Malignant cells are commonly characterised by being capable of invading tissue, growing self-sufficiently and uncontrollably, being insensitive to apoptosis induction and controlling their environment, for example inducing angiogenesis. Amongst them, a subpopulation of cancer cells, called cancer stem cells (CSCs) shows sustained replicative potential, tumor-initiating properties and chemoresistance. These characteristics make CSCs responsible for therapy resistance, tumor relapse and growth in distant organs, causing metastatic dissemination. For these reasons, eliminating CSCs is necessary in order to achieve long-term survival of cancer patients. New insights in cancer metabolism have revealed that cellular metabolism in tumors is highly heterogeneous and that CSCs show specific metabolic traits supporting their unique functionality. Indeed, CSCs adapt differently to the deprivation of specific nutrients that represent potentially targetable vulnerabilities. This review focuses on three of the most aggressive tumor types: pancreatic ductal adenocarcinoma (PDAC), hepatocellular carcinoma (HCC) and glioblastoma (GBM). The aim is to prove whether CSCs from different tumour types share common metabolic requirements and responses to nutrient starvation, by outlining the diverse roles of glucose and amino acids within tumour cells and in the tumour microenvironment, as well as the consequences of their deprivation. Beyond their role in biosynthesis, they serve as energy sources and help maintain redox balance. In addition, glucose and amino acid derivatives contribute to immune responses linked to tumourigenesis and metastasis. Furthermore, potential metabolic liabilities are identified and discussed as targets for therapeutic intervention.

Quality of Life in European Older Adults of SHARE Wave 7: Comparing the Old and the Oldest-Old.

CASP-12 (Control, Autonomy, Self-realization, and Pleasure scale) is one of the most common internationally used measures for quality of life in older adults, although its structure is not clearly established. Current research aims to test the factor structure of the CASP-12, so as to provide evidence on reliability and external validity, and to test for measurement invariance across age groups. Data from 61,355 Europeans (≥60 years old) from the Survey of Health, Ageing and Retirement in Europe wave 7 were used. CASP-12, EURO-D (European depression scale), self-perceived health, and life satisfaction measurements were included. Reliability and validity coefficients, competing confirmatory factor models, and standard measurement invariance routine were estimated. A second-order factor model with the original factor structure was retained. The scale showed adequate reliability coefficients except for the autonomy dimension. The correlation coefficients for external validity were all statistically significant. Finally, CASP-12 is scalar invariant across age. We conclude that the best-fitting factor structure retained allows using CASP-12 either by factors, or as an overall score, depending on the research interests. Findings related to CASP-12 measurement invariance encourage its use in the oldest-old too. When comparing the dimensions across age groups, as people age, autonomy slightly increases and the rest of the dimensions decline.

Inhibition of Mitochondrial Dynamics Preferentially Targets Pancreatic Cancer Cells with Enhanced Tumorigenic and Invasive Potential.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors, partly due to its intrinsic aggressiveness, metastatic potential, and chemoresistance of the contained cancer stem cells (CSCs). Pancreatic CSCs strongly rely on mitochondrial metabolism to maintain their stemness, therefore representing a putative target for their elimination. Since mitochondrial homeostasis depends on the tightly controlled balance between fusion and fission processes, namely mitochondrial dynamics, we aim to study this mechanism in the context of stemness. In human PDAC tissues, the mitochondrial fission gene DNM1L (DRP1) was overexpressed and positively correlated with the stemness signature. Moreover, we observe that primary human CSCs display smaller mitochondria and a higher DRP1/MFN2 expression ratio, indicating the activation of the mitochondrial fission. Interestingly, treatment with the DRP1 inhibitor mDivi-1 induced dose-dependent apoptosis, especially in CD133+ CSCs, due to the accumulation of dysfunctional mitochondria and the subsequent energy crisis in this subpopulation. Mechanistically, mDivi-1 inhibited stemness-related features, such as self-renewal, tumorigenicity, and invasiveness and chemosensitized the cells to the cytotoxic effects of Gemcitabine. In summary, mitochondrial fission is an essential process for pancreatic CSCs and represents an attractive target for designing novel multimodal treatments that will more efficiently eliminate cells with high tumorigenic potential.

Medición de la voluntad de vivir en adultos mayores: adaptación transcultural, validez y confiabilidad de la Will-to-Live Scale / Measurement of the will to live in older adults: Transcultural adaptation, validity and reliability of the Will-to-Live Scale

OBJETIVO: La Will-to-Live Scale (WTLS) es utilizada para medir la voluntad de vivir en adultos mayores; sin embargo, no existe una versión en español. El objetivo del estudio fue traducir la WTLS al español, evaluar su estructura interna, su confiabilidad y los correlatos entre la WTLS y la satisfacción con la vida, la resiliencia y la depresión en adultos mayores peruanos. MÉTODO: Los participantes fueron 235 adultos mayores peruanos (M=72,69, DE=6,68) evaluados entre marzo y mayo de 2019, seleccionados a través de un muestreo no probabilístico. Se administró la WTLS, la Satisfaction with Life Scale (SWLS), la Brief Resilient Coping Scale (BRCS) y el Patient Health Questionnarie-2 (PHQ-2). El análisis de datos incluyó confiabilidad por consistencia interna y modelos de ecuaciones estructurales, específicamente el análisis factorial confirmatorio (AFC), para probar la solución unidimensional de la WTLS y la validez convergente de la WTLS a nivel latente, al especificar un modelo de cuatro factores (voluntad de vivir, satisfacción con la vida, resiliencia y depresión). RESULTADOS: El coeficiente alfa de Cronbach y el índice de confiabilidad compuesto obtienen valores de 0,93 y 0,94, respectivamente. La estructura unidimensional de la WTLS se ajustó a los datos (χ2(5)=10,067, p = 0,073, CFI=0,999, RMSEA=0,066, SRMR=0,014) y mostró asociaciones positivas con la SWLS (ρ=0,82) y la BRCS (ρ=0,86), así como negativa con el PHQ-2 (ρ=−0,66). CONCLUSIÓN: La WTLS en español presenta evidencias de validez y confiabilidad para medir la voluntad de vivir en adultos mayores peruanos

OBJECTIVE: The Will-to-Live Scale (WTLS) is used to measure the will to live in older adults; however, there is no Spanish version. The objective of the study was to translate the WTLS into Spanish, assess its internal structure, reliability, and the correlates between WTLS and life satisfaction, resilience, and depression in older Peruvian adults. METHOD: The participants were 235 Peruvian older adults (M=72.69, SD=6.68), evaluated between March to May 2019, selected through non-probability sampling. The WTLS, the Satisfaction with Life Scale (SWLS), the Brief Resilient Coping Scale (BRCS) and the Patient Health Questionnaire-2 (PHQ-2) were administered. Data analysis included reliability by internal consistency and structural equation models, specifically confirmatory factor analysis (AFC), to test the one-dimensional solution of the WTLS and the convergent validity of the WTLS at the latent level, by specifying a four-factor model (will to live, life satisfaction, resilience and depression). RESULTS: Cronbach's alpha coefficient and the composite reliability index obtain values of .93 and .94, respectively. The one-dimensional structure of the WTLS was fitted to the data (χ2(5)=10,067, P=.073, CFI=.999, RMSEA=.066, SRMR=.014) and showed positive associations with the SWLS (ρ=.82), and BRCS (ρ=.86), as well as negative associations with the PHQ-2 (ρ=−.66). CONCLUSION: The WTLS in Spanish presents evidence of validity and reliability to measure the will to live in Peruvian older adults

Measurement of the will to live in older adults: Transcultural adaptation, validity and reliability of the Will-to-Live Scale. / Medición de la voluntad de vivir en adultos mayores: adaptación transcultural, validez y confiabilidad de la Will-to-Live Scale.

OBJECTIVE: The Will-to-Live Scale (WTLS) is used to measure the will to live in older adults; however, there is no Spanish version. The objective of the study was to translate the WTLS into Spanish, assess its internal structure, reliability, and the correlates between WTLS and life satisfaction, resilience, and depression in older Peruvian adults. METHOD: The participants were 235 Peruvian older adults (M=72.69, SD=6.68), evaluated between March to May 2019, selected through non-probability sampling. The WTLS, the Satisfaction with Life Scale (SWLS), the Brief Resilient Coping Scale (BRCS) and the Patient Health Questionnaire-2 (PHQ-2) were administered. Data analysis included reliability by internal consistency and structural equation models, specifically confirmatory factor analysis (AFC), to test the one-dimensional solution of the WTLS and the convergent validity of the WTLS at the latent level, by specifying a four-factor model (will to live, life satisfaction, resilience and depression). RESULTS: Cronbach's alpha coefficient and the composite reliability index obtain values of .93 and .94, respectively. The one-dimensional structure of the WTLS was fitted to the data (χ2(5)=10,067, P=.073, CFI=.999, RMSEA=.066, SRMR=.014) and showed positive associations with the SWLS (ρ=.82), and BRCS (ρ=.86), as well as negative associations with the PHQ-2 (ρ=-.66). CONCLUSION: The WTLS in Spanish presents evidence of validity and reliability to measure the will to live in Peruvian older adults.

Molecular and Metabolic Subtypes Correspondence for Pancreatic Ductal Adenocarcinoma Classification.

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is an extremely lethal disease due to late diagnosis, aggressiveness and lack of effective therapies. Considering its intrinsic heterogeneity, patient stratification models based on transcriptomic and genomic signatures, with partially overlapping subgroups, have been established. Besides molecular alterations, PDAC tumours show a strong desmoplastic response, resulting in profound metabolic reprogramming involving increased glucose and amino acid consumption, as well as lipid scavenging and biosynthesis. Interestingly, recent works have also revealed the existence of metabolic subtypes with differential prognosis within PDAC, which correlated to defined molecular subclasses in patients: lipogenic subtype correlated with a classical/progenitor signature, while glycolytic tumours associated with the highly aggressive basal/squamous profile. Bioinformatic analyses have demonstrated that the representative genes of each metabolic subtype are up-regulated in PDAC samples and predict patient survival. This suggests a relationship between the genetic signature, metabolic profile, and aggressiveness of the tumour. Considering all this, defining metabolic subtypes represents a clear opportunity for patient stratification considering tumour functional behaviour independently of their mutational background.

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.

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.

Erratum: Pérez-Belmonte, S., et al. Subtypes of Depression: Latent Class Analysis in Spanish Old People with Depressive Symptoms. Life 2020, 10, 70.

The authors wish to make the following erratum to this paper [...].