Auto-fluorescent intracellular sink - A novel Inherent biomarker for drug discovery in pancreatic cancer stem cells

Tesis doctoral inédita, leída en Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 20/09/2013.Pancreatic adenocarcinoma (PDAC), the fourth leading cause of cancer-­‐ related death world-­‐wide, is a malignant neoplasm of the exocrine pancreas. It has been hypothesized that a subset of tumor cells with stem-­‐like properties, termed cancer stem cells (CSCs), drives pancreatic tumor growth, metastasis, and chemoresistance. While multiple surface markers such as CD133 and CD44 have been successfully used to isolate and characterize CSCs, their expression are not exclusively linked to a CSC functional phenotype. Therefore, since isolating and characterizing CSCs is of paramount importance for understanding pancreatic cancer, we sought to identify new and novel markers that functionally enrich for CSCs using primary human pancreatic cancer cells. While our classic approaches, such as cell surface expression of known CSC markers or side population were either prone to alterations by the tissue culture environment or did not enrich for CSCs, we inadvertently identified a distinct population of cells characterized by a subcellular compartment exhibiting strong autofluorescence, which did exhibit defined CSC characteristics. Specifically, these autofluorescent cells were markedly enriched both in sphere culture and during chemotherapy, strongly expressed pluripotency-­‐associated genes, and were highly invasive both in vitro and in vivo. Most importantly, they were exclusively tumorigenic in vivo at the single cell level and were capable of recapitulating the heterogeneity of the parental tumor. Autofluorescence was determined to be due to an accumulation of riboflavin in membrane-­‐restricted cytoplasmic structures by means of the ATP-­‐dependent ABCG2 transporter, which did not overlap with cells defined as the side population, but could be selectively eliminated with the ABCG2 inhibitor Fumitremorgin C. In addition, we show that autofluorescence is not restricted to PDAC, as other solid tumors similarly contain autofluorescent cells. Thus, to take advantage of this broad phenotype, we developed a low throughput screening platform, and show that autofluorescent cells are highly amendable to anti-­‐cancer compound screening. Thus, unbiased and label-­‐free tracking of autofluorescent cells in cancers such as PDAC represents a promising new technological advancement, which can be used not only for isolating and studying CSC, but can be additionally exploited for low throughput screening of compounds with anti-­‐cancer activity in a clinical setting.El adenocarcinoma pancreático es una neoplasia maligna del páncreas exocrino, siendo la cuarta causa principal de muerte relacionada con cáncer en todo el mundo. Se ha planteado la hipótesis de que un subconjunto de células tumorales con propiedades troncales, denominadas células madre de cáncer, impulsan el crecimiento del tumor de páncreas, metástasis, y la quimiorresistencia. Mientras que varios marcadores de superficie tales como CD133 y CD44 se han utilizado con éxito para aislar y caracterizar este tipo de células, su expresión no está vinculada exclusivamente a un fenotipo funcional de células madre de cáncer. Por tanto, ya que el aislamiento y caracterización de este tipo de células es de vital importancia, hemos tratado de identificar marcadores novedosos que de manera funcional, enriquezan en este tipo celular utilizando células primarias de cáncer de páncreas de humanos. Los resultados premilinares de la búsqueda de un buen marcador de células madre de cáncer, tales como la expresión de marcadores de superficie o las células Side-­‐Population (SP), observamos que podían estar sujetos a alteraciones por el cultivo de tejidos o que no enriquencían de manera especifíca en células madre. Esto nos forzó a la búsqueda de otro marcador que no fuse sensible a este tipo de alteraciones en su expresión, siendo éste nuestro principial objetivo de este trabajo. Partiendo de cultivos primarios de xenoinjertos humanos, identificamos una población de células que se caracteriza por un compartimento subcelular exhibiendo una fuerte autofluorescencia. Específicamente, estas células autofluorescentes fueron enriquecidas tanto en cultivo de esferas como durante la quimioterapia, sobre-­‐expresan los genes de pluripotencia-­‐asociados y mostraron ser altamente invasivas, tanto in vitro como in vivo. Además, estas celulas son exclusivamente tumorigénicas in vivo, siendo capaces de formar un tumor desde tan sólo una célula. Se determinó que la autofluorescencia era una acumulación de riboflavina en la estructura autofluorescente y estaba mediado por el transportador ABCG2. Debido a que se expresan en una gran parte de tumores primarios de páncreas y su expresión se mantiene constante bajo diferentes condiciones, desarrollamos una plataforma de screening donde podemos investigar el efecto de distintos compuestos anticancerígenos, así como obtener un diagnóstico clinico para medicina personalizada

Development of a purification process for HIV-1 VLPs, from supernatant to lyophilization

HIV-1-based virus-like particles (VLPs) have high potential as scaffold for the development of chimeric or multivalent vaccines by functionalizing them with specific antigens [1], [2]. The obtention of vaccine formulations independent of cold chain is desirable to facilitate transportation and administration worldwide [3]. Recently, efforts are being made to develop cost-effective scalable processes to obtain these particles. The present study aims to compare some of the most used downstream processing (DSP) technologies for capture and purification of VLPs, taking especial consideration in using technologies enabling operation at large scale [4]. First, suspension adapted HEK 293 cells cultured in chemically defined cell culture media were used to produce the Gag-eGFP VLPs. Then, some steps of the purification process were studied, including a primary and secondary clarification by depth filtration and filtration respectively, an intermediate step by tangential flow filtration (TFF) or multimodal chromatography (MC), a capture step by ion exchange (IEC), heparin affinity (AC) and hydrophobic interaction chromatography (HIC), a polishing step by size exclusion chromatography (SEC) and a finally lyophilization step by freeze-drying process. Different operation units were tested for each step. Finally, a complete DSP train was implemented using the best results obtained in each stage. A concentration of 2.2 ± 0.8·109 VLPs/mL in the lyophilized samples was obtained after its storage at room temperature for 2 months. The morphology and structural integrity were further assessed by cryo-TEM. These first results in enveloped VLP lyophilization offer great promise to overcome the difficulty to distribute vaccines in poorly served remote rural areas and increase vaccine stability until their administration. Likewise, the purification methodologies proposed here could be easily scaled up and applied to purify similar enveloped viruses and vesicles. Please click Additional File below for the presentation

The miR-17-92 cluster counteracts quiescence and chemoresistance in a distinct subpopulation of pancreatic cancer stem cells

Objective Cancer stem cells (CSCs) represent the root of many solid cancers including pancreatic ductal adenocarcinoma, are highly chemoresistant and represent the cellular source for disease relapse. However the mechanisms involved in these processes still need to be fully elucidated. Understanding the mechanisms implicated in chemoresistance and metastasis of pancreatic cancer is critical to improving patient outcomes. Design Micro-RNA (miRNA) expression analyses were performed to identify functionally defining epigenetic signatures in pancreatic CSC-enriched sphere-derived cells and gemcitabine-resistant pancreatic CSCs. Results We found the miR-17-92 cluster to be downregulated in chemoresistant CSCs versus non-CSCs and demonstrate its crucial relevance for CSC biology. In particular, overexpression of miR-17-92 reduced CSC self-renewal capacity, in vivo tumourigenicity and chemoresistance by targeting multiple NODAL/ACTIVIN/TGF-beta 1 signalling cascade members as well as directly inhibiting the downstream targets p21, p57 and TBX3. Overexpression of miR-17-92 translated into increased CSC proliferation and their eventual exhaustion via downregulation of p21 and p57. Finally, the translational impact of our findings could be confirmed in preclinical models for pancreatic cancer. Conclusions Our findings therefore identify the miR-17-92 cluster as a functionally determining family of miRNAs in CSCs, and highlight the putative potential of developing modulators of this cluster to overcome drug resistance in pancreatic CSCs.CH: ERC Advanced Investigator Grant (Pa-CSC 233460), European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement No 256974 (EPC-TM-NET) and No 602783 (CAM-PaC), the Subdireccion General de Evaluacion y Fomento de la Investigacion, Fondo de Investigacion Sanitaria (PS09/02129 \& PI12/02643), and the Programa Nacional de Internacionalizacion de la I+D, Subprogramma: FCCI 2009 (PLE2009-0105; Ministerio de Economia y Competitividad, Spain). MC: La Caixa Predoctoral Fellowship.S

Intracellular autofluorescence as a new biomarker for cancer stem cells in glioblastoma

The identification of cancer stem cells (CSCs), which are implicated in tumor initiation, progression, therapy resistance, and relapse, is of great biological and clinical relevance. In glioblastoma (GBM), this is still a challenge, as no single marker is able to universally identify populations of GBM cancer stem cells (GSCs). Indeed, there is still controversy on whether biomarker-expressing cells fulfill the functional criteria of bona fide GSCs, despite being widely used. Here, we describe a novel subpopulation of autofluorescent (Fluo+) cells in GBM that bear all the functional characteristics of GSCs, including higher capacity to grow as neurospheres, long-term self-renewal ability, increased expression of stem cell markers, and enhanced in vivo tumorigenicity. Mechanistically, the autofluorescent phenotype is largely due to the intracellular accumulation of riboflavin, mediated by the ABC transporter ABCG2. In summary, our work identifies an intrinsic cellular autofluorescent phenotype enriched in GBM cells with functional stem cells features that can be used as a novel, simple and reliable biomarker to target these highly malignant tumors, with implications for GBM biological and clinical research.This research was funded by FEDER funds through the Operational Programme Competitiveness Factors–COMPETE and National Funds through FCT under the projects UIDB/50026/2020, UIDP/50026/2020, and POCI-01-0145-FEDER-007038; by the project NORTE-01-0145-FEDER-000013, NORTE-01-0246-FEDER-000012, and NORTE-01-0145-FEDER-000023, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). J.V.d.C., C.S.G., E.P.M., and B.M.C. was funded by FCT-Foundation for Science and Technology (SFRH/BD/88121/2012 to J.V.d.C.; SFRH/BD/92786/2013 to C.S.G.; PD/BDE/143154/2019 to E.P.M.; and PTDC/SAUGMG/113795/2009, IF/00601/2012 and CEECIND/00072/2018 to B.M.C.). B.M.C. was also funded by Fundação Calouste Gulbenkian and Liga Portuguesa Contra o Cancro

Microenvironmental hCAP-18/LL-37 promotes pancreatic ductal adenocarcinoma by activating its cancer stem cell compartment

This is the peer reviewed version of the following article: Microenvironmental hCAP-18/LL-37 promotes pancreatic ductal adenocarcinoma by activating its cancer stem cell compartment. Gut 64.12 (2015): 1921-1935 and which has been published in final form at http://dx.doi.org/10.1136/gutjnl-2014-308935OBJECTIVES: The tumour stroma/microenvironment not only provides structural support for tumour development, but more importantly it provides cues to cancer stem cells (CSCs) that regulate their self-renewal and metastatic potential. This is certainly true for pancreatic ductal adenocarcinomas (PDAC), where tumour-associated fibroblasts, pancreatic stellate cells and immune cells create an abundant paracrine niche for CSCs via microenvironment-secreted factors. Thus understanding the role that tumour stroma cells play in PDAC development and CSC biology is of utmost importance. DESIGN: Microarray analyses, tumour microarray immunohistochemical assays, in vitro co-culture experiments, recombinant protein treatment approaches and in vivo intervention studies were performed to understand the role that the immunomodulatory cationic antimicrobial peptide 18/LL-37 (hCAP-18/LL-37) plays in PDAC biology. RESULTS: We found that hCAP-18/LL-37 was strongly expressed in the stroma of advanced primary and secondary PDAC tumours and is secreted by immune cells of the stroma (eg, tumour-associated macrophages) in response to tumour growth factor-β1 and particularly CSC-secreted Nodal/ActivinA. Treatment of pancreatic CSCs with recombinant LL-37 increased pluripotency-associated gene expression, self-renewal, invasion and tumourigenicity via formyl peptide receptor 2 (FPR2)- and P2X purinoceptor 7 receptor (P2X7R)-dependent mechanisms, which could be reversed by inhibiting these receptors. Importantly, in a genetically engineered mouse model of K-Ras-driven pancreatic tumourigenesis, we also showed that tumour formation was inhibited by either reconstituting these mice with bone marrow from cathelicidin-related antimicrobial peptide (ie, murine homologue of hCAP-18/LL-37) knockout mice or by pharmacologically inhibiting FPR2 and P2X7R. CONCLUSIONS: Thus, hCAP-18/LL-37 represents a previously unrecognised PDAC microenvironment factor that plays a critical role in pancreatic CSC-mediated tumourigenesis.CH: ERC Advanced Investigator Grant (Pa-CSC 233460), European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 256974 (EPC-TM-NET) and n° 602783 (CAM-PaC), the Subdirección General de Evaluación y Fomento de la Investigación, Fondo de Investigación Sanitaria (PS09/02129 & PI12/02643) and the Programa Nacional de Internacionalización de la I+D, Subprogramma: FCCI 2009 (PLE2009-0105; both Ministerio de Economía y Competitividad (es), Spain), BSJr: Rámon y Cajal Merit Award from the Ministerio de Economía y Competitividad, Spain and Clinic and Laboratory Integration Program (CLIP) grant from the Cancer Research Institute, NY, NY. MC: La Caixa Predoctoral Fellowshi

Osteoprotegerin in Exosome-Like Vesicles from Human Cultured Tubular Cells and Urine

Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and receptors may be present in exosomes and are expressed by proximal tubular cells. We have now studied the expression of selected TNF superfamily proteins in exosome-like vesicles from cultured human proximal tubular cells and human urine and have identified additional proteins in these vesicles by LC-MS/MS proteomics. Human proximal tubular cells constitutively released exosome-like vesicles that did not contain the TNF superfamily cytokines TRAIL or TWEAK. However, exosome-like vesicles contained osteoprotegerin (OPG), a TNF receptor superfamily protein, as assessed by Western blot, ELISA or selected reaction monitoring by nLC-(QQQ)MS/MS. Twenty-one additional proteins were identified in tubular cell exosomelike vesicles, including one (vitamin D binding protein) that had not been previously reported in exosome-like vesicles. Twelve were extracellular matrix proteins, including the basement membrane proteins type IV collagen, nidogen-1, agrin and fibulin-1. Urine from chronic kidney disease patients contained a higher amount of exosomal protein and exosomal OPG than urine from healthy volunteers. Specifically OPG was increased in autosomal dominant polycystic kidney disease urinary exosome-like vesicles and expressed by cystic epithelium in vivo. In conclusion, OPG is present in exosome-like vesicles secreted by proximal tubular epithelial cells and isolated from Chronic Kidney Disease urine.This work was supported by grants from the Instituto de Salud Carlos III (ISCIIIRETIC REDINREN RD06/0016, RD12/0021, PI11/01854, PI10/00072 PI09/ 00641 and PS09/00447); Comunidad de Madrid (Fibroteam S2010/BMD-2321, S2010/BMD-2378); Sociedad Española de NefrologÍa; European Network (HEALTH F2-2008-200647); DIALOK European project LSHB-CT-2007-036644; Fundacion Lilly and IRSIN/FRIAT to JE; Programa Intensificación Actividad Investigadora (ISCIII/ Agencia Laín-Entralgo/CM) to AO; Instituto de Salud Carlos III (FIS PI11/01401, CP09/00229); and Fundación Conchita Rábago de Jiménez DÍaz to GAL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Famílies botàniques de plantes medicinals

Facultat de Farmàcia, Universitat de Barcelona. Ensenyament: Grau de Farmàcia, Assignatura: Botànica Farmacèutica, Curs: 2013-2014, Coordinadors: Joan Simon, Cèsar Blanché i Maria Bosch.Els materials que aquí es presenten són els recull de 175 treballs d’una família botànica d’interès medicinal realitzats de manera individual. Els treballs han estat realitzat per la totalitat dels estudiants dels grups M-2 i M-3 de l’assignatura Botànica Farmacèutica durant els mesos d’abril i maig del curs 2013-14. Tots els treballs s’han dut a terme a través de la plataforma de GoogleDocs i han estat tutoritzats pel professor de l’assignatura i revisats i finalment co-avaluats entre els propis estudiants. L’objectiu principal de l’activitat ha estat fomentar l’aprenentatge autònom i col·laboratiu en Botànica farmacèutica

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab

The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension