Análisis de la metástasis del cáncer colorectal mediante proteómica y microscopía

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, leída el 08-11-2022Cancer is the second most common cause of death, only preceded by cardiovascular diseases. The main reason behind the high mortality rates of cancer is the appearance of metastasis, the dissemination and colonisation of secondary tissues by cells from the primary tumour. In particular, in the case of colorectal cancer (CRC) the survival rates of patients diagnosed, drop drastically if diagnosis happens at later stages when metastasis has occurred. A similar trend can be observed for most of the cancers commonly diagnosed. When metastasis occurs, treatment relies heavily on the use of chemotherapy, even if the original tumour mass is surgically removed, the metastases will still survive. It is thus paramount to find new tools that improve early diagnosis so that detection can occur before the tumour spreads. In this context, the main focus of this thesis has been the understanding and characterisation of CRC metastasis to find new diagnostic markers that can be used in the clinic. We have used isogenic cell lines, that share the same genetic background but have different metastatic capacities, to define the proteome of CRC metastasis in vitro...Tras las enfermedades cardiovasculares, la segunda causa de muerte en países desarrollados es el cáncer. El principal factor detrás de la alta mortalidad del cáncer es la aparición de metástasis, el proceso por el cual células provenientes de la masa tumoral original diseminan y son capaces de colonizar tejidos distantes. En el caso del cáncer colorrectal(CCR), las tasas de supervivencia de los pacientes caen de manera drástica si el diagnóstico de la enfermedad se produce en estadios tardíos en los que ya haya aparecido metástasis. Esta tendencia se puede observar también en la mayoría de los cánceres diagnosticados en la actualidad. Uno de los principales problemas de la metástasis es que una vez aparece, el tratamiento del cáncer se vuelve complejo, siendo la quimioterapia la principal forma de tratamiento ya que, aunque se elimine quirúrgicamente la masa tumoral original, los nichos metastáticos sobrevivirán a la operación. Es por tanto necesario el desarrollo de nuevas técnicas de diagnóstico que permitan mejorar la detección temprana, antes de que el tumor se extienda. En este contexto, el principal objetivo de esta tesis ha sido entender y caracterizar la metástasis del CCR para encontrar marcadores de diagnóstico que puedan trasladarse a la práctica clínica. Para definir el proteoma de la metástasis de CCR hemos empleado una serie de líneas celulares isogénicas, que comparten la misma carga genética, con diferente potencial metastático...Fac. de Ciencias QuímicasTRUEunpu

Metabolic Reprogramming Helps to Define Different Metastatic Tropisms in Colorectal Cancer

Approximately 25% of colorectal cancer (CRC) patients experience systemic metastases, with the most frequent target organs being the liver and lung. Metabolic reprogramming has been recognized as one of the hallmarks of cancer. Here, metabolic and functional differences between two CRC cells with different metastatic organotropisms (metastatic KM12SM CRC cells to the liver and KM12L4a to the lung when injected in the spleen and in the tail vein of mice) were analysed in comparison to their parental non-metastatic isogenic KM12C cells, for a subsequent investigation of identified metabolic targets in CRC patients. Meta-analysis from proteomic and transcriptomic data deposited in databases, qPCR, WB, in vitro cell-based assays, and in vivo experiments were used to survey for metabolic alterations contributing to their different organotropism and for the subsequent analysis of identified metabolic markers in CRC patients. Although no changes in cell proliferation were observed between metastatic cells, KM12SM cells were highly dependent on oxidative phosphorylation at mitochondria, whereas KM12L4a cells were characterized by being more energetically efficient with lower basal respiration levels and a better redox management. Lipid metabolism-related targets were found altered in both cell lines, including LDLR, CD36, FABP4, SCD, AGPAT1, and FASN, which were also associated with the prognosis of CRC patients. Moreover, CD36 association with lung metastatic tropism of CRC cells was validated in vivo. Altogether, our results suggest that LDLR, CD36, FABP4, SCD, FASN, LPL, and APOA1 metabolic targets are associated with CRC metastatic tropism to the liver or lung. These features exemplify specific metabolic adaptations for invasive cancer cells which stem at the primary tumour.This work was supported by grants cofounded by Fondo Europeo de Desarrollo Regional -FEDER- PI17CIII/00045 and PI20CIII/00019 from the AES-ISCIII program to RB from the Instituto de Salud Carlos III (ISCIII) and grants from Spanish Ministry of Science (Plan Nacional I+D+i PID2019-110183RBC21), Regional Government of Community of Madrid (P2018/BAA-4343-ALIBIRD2020-CM, and Y2020/BIO-6350), and Ramón Areces Foundation (CIVP19A5937) to AR. AM-C FPU predoctoral contract is supported by the Spanish Ministerio de Educació n, Cultura y Deporte. AQ-F acknowledges Comunidad de Madrid for the Garantıa Juvenil PEJD-2017-RE/BMD-3394 contract. GS-F is a recipient of a predoctoral contract (grant number 1193818N) supported by the Flanders Research Foundation (FWO).S

Phase II clinical trial of nab-paclitaxel plus gemcitabine in elderly patients with previously untreated locally advanced or metastatic pancreatic adenocarcinoma: the BIBABRAX study

[Purpose] To evaluate the health-related quality of life (HRQoL), global health status (GHS), and deterioration-free survival of an elderly population (> 70 years) with unresectable locally advanced (LAPC) or metastatic pancreatic cancer (mPC) treated with nab-paclitaxel in combination with gemcitabine.[Methods] In this open-label, single-arm, multicenter, phase II trial, patients received 4-week cycles of intravenous (i.v.) nab-paclitaxel at a dose of 125 mg/m2, followed by i.v. injections of gemcitabine at a dose of 1000 mg/m2 on days 1, 8 and 15 until disease progression or unacceptable toxicity was observed. The primary outcome was the HRQoL (deterioration-free rate at 3 months as evaluated with the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30.[Results] Eighty patients (median age: 74.6 years) were enrolled (56 with mPC, 24 with LAPC). The percentage of patients who had not experienced deterioration at 3 months was 54.3% (95% CI 41.6–67.0%). The median (interquartile range) time until definite deterioration was 1.6 (1.1–3.7) months. The objective response rate and clinical benefit rate were achieved by 11 (13.8%, 95% CI 6.2–21.3%) and 54 patients (67.5%, 95% CI 57.2–77.8%), respectively. The median overall survival was 9.2 months (95% CI 6.9–11.5), and the median progression-free survival was 7.2 months (95% CI 5.8–8.5). Only fatigue and neutropenia demonstrated a grade 3–4 toxicity incidence > 20%.[Conclusions] Our study confirms the clinical benefit of the combination of nab-paclitaxel and gemcitabine in an elderly population with pancreatic cancer in terms of improved survival and clinical response. However, we were unable to confirm a benefit in terms of quality-of-life.The BIBABRAX study was funded by Celgene. Medical writing assistance in the preparation of this paper was provided by Apices with financial support from Celgene.Peer reviewe

Resistance to Bleomycin-Induced Lung Fibrosis in MMP-8 Deficient Mice Is Mediated by Interleukin-10

BACKGROUND: Matrix metalloproteinases (MMPs) may have pro and antifibrotic roles within the lungs, due to its ability to modulate collagen turnover and immune mediators. MMP-8 is a collagenase that also cleaves a number of cytokines and chemokines. METHODOLOGY AND PRINCIPAL FINDINGS: To evaluate its relevance in lung fibrosis, wildtype and Mmp8(-/-) mice were treated with either intratracheal bleomycin or saline, and lungs were harvested at different time points. Fibrosis, collagen, collagenases, gelatinases, TGFβ and IL-10 were measured in lung tissue. Mmp8(-/-) mice developed less fibrosis than their wildtype counterparts. This was related to an increase in lung inflammatory cells, MMP-9 and IL-10 levels in these mutant animals. In vitro experiments showed that MMP-8 cleaves murine and human IL-10, and tissue from knockout animals showed decreased IL-10 processing. Additionally, lung fibroblasts from these mice were cultured in the presence of bleomycin and collagen, IL-10 and STAT3 activation (downstream signal in response to IL-10) measured by western blotting. In cell cultures, bleomycin increased collagen synthesis only in wildtype mice. Fibroblasts from knockout mice did not show increased collagen synthesis, but increased levels of unprocessed IL-10 and STAT3 phosphorylation. Blockade of IL-10 reverted this phenotype, increasing collagen in cultures. CONCLUSIONS: According to these results, we conclude that the absence of MMP-8 has an antifibrotic effect by increasing IL-10 and propose that this metalloprotease could be a relevant modulator of IL-10 metabolism in vivo

Phage-Derived and Aberrant HaloTag Peptides Immobilized on Magnetic Microbeads for Amperometric Biosensing of Serum Autoantibodies and Alzheimer's Disease Diagnosis

An electrochemical biosensing platform for serum autoantibodies (AAbs) detection is reported in this work, exploiting for the first time six Alzheimer's disease (AD)-specific phage-derived and frameshift aberrant HaloTag peptides as receptors, immobilized on magnetic microbeads (MBs) surface and captured on disposable electrodes to perform amperometric detection. Operational analytical characteristics and clinical diagnostic ability of the bioplatform were probed in optimized key experimental conditions by analysing serum AAbs of AD patients and healthy subjects. The value of 100% obtained for AUC, sensitivity, and selectivity from the all peptides combined ROC curve, indicate full AD-diagnostic capability of the methodology, which was further implemented, as proof of concept, in a POC multiplexing platform to detect the signature in a single test over clinically actionable times (1 h 15 min), opening great promise for the type of diagnosis and AD patients’ monitoring follow-up currently pursued

Aryl-hydrocarbon receptor-interacting protein regulates tumorigenic and metastatic properties of colorectal cancer cells driving liver metastasis

12 p.-5 fig.-1 tab.Background: Liver metastasis is the primary cause of colorectal cancer (CRC)-associated death. Aryl-hydrocarbon receptor-interacting protein (AIP), a putative positive intermediary in aryl-hydrocarbon receptor-mediated signalling, is overexpressed in highly metastatic human KM12SM CRC cells and other highly metastatic CRC cells.Methods: Meta-analysis and immunohistochemistry were used to assess the relevance of AIP. Cellular functions and signalling mechanisms mediated by AIP were assessed by gain-of-function experiments and in vitro and in vivo experiments.Results: A significant association of high AIP expression with poor CRC patients’ survival was observed. Gain-of-function and quantitative proteomics experiments demonstrated that AIP increased tumorigenic and metastatic properties of isogenic KM12C (poorly metastatic) and KM12SM (highly metastatic to the liver) CRC cells. AIP overexpression dysregulated epithelial-to-mesenchymal (EMT) markers and induced several transcription factors and Cadherin-17 activation. The former induced the signalling activation of AKT, SRC and JNK kinases to increase adhesion, migration and invasion of CRC cells. In vivo, AIP expressing KM12 cells induced tumour growth and liver metastasis. Furthermore, KM12C (poorly metastatic) cells ectopically expressing AIP became metastatic to the liver.Conclusions: Our data reveal new roles for AIP in regulating proteins associated with cancer and metastasis to induce tumorigenic and metastatic properties in colon cancer cells driving liver metastasis.This work was supported by the financial support of the PI17CIII/00045 and PI20CIII/00019 grants from the AES-ISCIII program to RB. J Hendrix acknowledges funding by UH-BOF (BOF20TT06). J Hofkens acknowledges financial support from the Research Foundation-Flanders (FWO, Grant No. ZW15_09-G0H6316N), the Flemish government through long-term structural funding Methusalem (CASAS2, Meth/15/04) and the MPI as MPI fellow. S.R. acknowledges the financial support of the KU Leuven through the internal C1 funding (KU Leuven (C14/16/053)). GSF is the recipient of a predoctoral contract (grant number 1193818 N) supported by The Flanders Research Foundation (FWO). The FPU predoctoral contract to AMC is supported by the Spanish Ministerio de Educación, Cultura y Deporte.Peer reviewe

MMP-8 Deficiency Increases TLR/RAGE Ligands S100A8 and S100A9 and Exacerbates Lung Inflammation during Endotoxemia

Matrix metalloproteinase-8, released mainly from neutrophils, is a critical regulator of the inflammatory response by its ability to cleave multiple mediators. Herein, we report the results of a model of endotoxemia after intraperitoneal LPS injection in mice lacking MMP-8 and their wildtype counterparts. Control, saline-treated animals showed no differences between genotypes. However, there was an increased lung inflammatory response, with a prominent neutrophilic infiltration in mutant animals after LPS treatment. Using a proteomic approach, we identify alarmins S100A8 and S100A9 as two of the main differences between genotypes. Mice lacking MMP-8 showed a significant increase in these two molecules in lung homogenates, but not in spleen and serum. Mice lacking MMP-8 also showed an increase in MIP-1α levels and a marked activation of the non-canonical NF-κB pathway, with no differences in CXC-chemokines such as MIP-2 or LIX. These results show that MMP-8 can modulate the levels of S100A8 and S100A9 and its absence promotes the lung inflammatory response during endotoxemia

Healthcare workers hospitalized due to COVID-19 have no higher risk of death than general population. Data from the Spanish SEMI-COVID-19 Registry

Aim To determine whether healthcare workers (HCW) hospitalized in Spain due to COVID-19 have a worse prognosis than non-healthcare workers (NHCW). Methods Observational cohort study based on the SEMI-COVID-19 Registry, a nationwide registry that collects sociodemographic, clinical, laboratory, and treatment data on patients hospitalised with COVID-19 in Spain. Patients aged 20-65 years were selected. A multivariate logistic regression model was performed to identify factors associated with mortality. Results As of 22 May 2020, 4393 patients were included, of whom 419 (9.5%) were HCW. Median (interquartile range) age of HCW was 52 (15) years and 62.4% were women. Prevalence of comorbidities and severe radiological findings upon admission were less frequent in HCW. There were no difference in need of respiratory support and admission to intensive care unit, but occurrence of sepsis and in-hospital mortality was lower in HCW (1.7% vs. 3.9%; p = 0.024 and 0.7% vs. 4.8%; p<0.001 respectively). Age, male sex and comorbidity, were independently associated with higher in-hospital mortality and healthcare working with lower mortality (OR 0.211, 95%CI 0.067-0.667, p = 0.008). 30-days survival was higher in HCW (0.968 vs. 0.851 p<0.001). Conclusions Hospitalized COVID-19 HCW had fewer comorbidities and a better prognosis than NHCW. Our results suggest that professional exposure to COVID-19 in HCW does not carry more clinical severity nor mortality

Modernización del Sistema Aduanero y su vinculación con la sociedad civil

Informe de investigación--Universidad de Costa Rica, Vicerrectoría de Acción Social, Trabajo Comunal Universitario. 2012. Para mayor información puede escribir a [email protected] documento es una guía a los agroexportadores para que conozcan información general sobre los formularios exigidos en el tema de exportación, tales como modernización del sistema aduanero costarricense, notificaciones previas, el compendio de leyes en materia aduanera, la inscripción como importador, entre otras. La información del presente documento se complementa con las presentaciones adjuntas en este fichero.Universidad de Costa RicaUCR::Vicerrectoría de Acción Social::Trabajo Comunal Universitario (TCU