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1.
EBioMedicine ; 46: 79-93, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31303496

RESUMEN

BACKGROUND: Metastatic colorectal cancer (CRC) remains a deadly disease. Identifying locally advanced CRC patients with high risk of developing metastasis and improving outcome of metastatic CRC patients require discovering master regulators of metastasis. In this context, the non-coding part of the human genome is still largely unexplored. METHODS: To interrogate the non-coding part of the human genome and disclose regulators of CRC metastasis, we combined a transposon-based forward genetic screen with a novel in vitro assay, which forces cells to grow deprived of cell-substrate and cell-cell contacts (i.e. forced single cell suspension assay - fSCS). FINDINGS: We proved that fSCS selects CRC cells with mesenchymal and pro-metastatic traits. Moreover, we found that the transposon insertions conferred CRC cells resistance to fSCS and thus metastatic advantage. Among the retrieved transposon insertions, we demonstrated that the one located in the 3'UTR of BTBD7 disrupts miR-23b::BTBD7 interaction and contributes to pro-metastatic traits. In addition, miR-23b and BTBD7 correlate with CRC metastasis both in preclinical experiments and in clinical samples. INTERPRETATION: fSCS is a simple and scalable in vitro assay to investigate pro-metastatic traits and transposon-based genetic screens can interrogate the non-coding part of the human genome (e.g. miRNA::target interactions). Finally, both Btbd7 and miR-23b represent promising prognostic biomarkers and therapeutic targets in CRC. FUND: This work was supported by Marie Curie Actions (CIG n. 303877) and Friuli Venezia Giulia region (Grant Agreement n°245574), Italian Association for Cancer Research (AIRC, MFAG n°13589), Italian Ministry of Health (GR-2010-2319387 and PE-2016-02361040) and 5x1000 to CRO Aviano.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Interferencia de ARN , Comunicación Celular , Línea Celular Tumoral , Proliferación Celular , Transición Epitelial-Mesenquimal/genética , Matriz Extracelular/metabolismo , Pruebas Genéticas , Humanos , Metástasis de la Neoplasia , Estadificación de Neoplasias
3.
Cancers (Basel) ; 10(8)2018 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-30110953

RESUMEN

Molecular characterization is currently a key step in NSCLC therapy selection. Circulating tumor cells (CTC) are excellent candidates for downstream analysis, but technology is still lagging behind. In this work, we show that the mutational status of NSCLC can be assessed on hypermetabolic CTC, detected by their increased glucose uptake. We validated the method in 30 Stage IV NSCLC patients: peripheral blood samples were incubated with a fluorescent glucose analog (2-NBDG) and analyzed by flow cytometry. Cells with the highest glucose uptake were sorted out. EGFR and KRAS mutations were detected by ddPCR. In sorted cells, mutated DNA was found in 85% of patients, finding an exact match with primary tumor in 70% of cases. Interestingly, in two patients multiple KRAS mutations were detected. Two patients displayed different mutations with respect to the primary tumor, and in two out of the four patients with a wild type primary tumor, new mutations were highlighted: EGFR p.746_750del and KRAS p.G12V. Hypermetabolic CTC can be enriched without the need of dedicated equipment and their mutational status can successfully be assessed by ddPCR. Finally, the finding of new mutations supports the possibility of probing tumor heterogeneity.

4.
Am J Transl Res ; 10(12): 4004-4016, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30662646

RESUMEN

In a recent paper we presented an innovative method of liquid biopsy, for the detection of circulating tumor cells (CTC) in the peripheral blood. Using microfluidics, CTC are individually encapsulated in water-in-oil droplets and selected by their increased rate of extracellular acidification (ECAR). During the analysis, empty or debris-containing droplets are discarded manually by screening images of positive droplets, increasing the operator-dependency and time-consumption of the assay. In this work, we addressed the limitations of the current method integrating computer vision techniques in the analysis. We implemented an automatic classification of droplets using convolutional neural networks, correctly classifying more than 96% of droplets. A second limitation of the technique is that ECAR is computed using an average droplet volume, without considering small variations in extracellular volume which can occur due to the normal variability in the size of the droplets or cells. Here, with the use of neural networks for object detection, we segmented the images of droplets and cells to measure their relative volumes, correcting over- or under-estimation of ECAR, which was present up to 20%. Finally, we evaluated whether droplet images contained additional information. We preliminarily gave a proof-of-concept demonstration showing that white blood cells expression of CD45 can be predicted with 82.9% accuracy, based on bright-field cell images alone. Then, we applied the method to classify acid droplets as coming from metastatic breast cancer patients or healthy donors, obtaining an accuracy of 90.2%.

5.
Int J Mol Sci ; 17(10)2016 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-27783057

RESUMEN

Although the enumeration of circulating tumor cells (CTC) defined as expressing both epithelial cell adhesion molecule and cytokeratins (EpCAM⁺/CK⁺) can predict prognosis and response to therapy in metastatic breast, colon and prostate cancer, its clinical utility (i.e., the ability to improve patient outcome by guiding therapy) has not yet been proven in clinical trials. Therefore, scientists are now focusing on the molecular characterization of CTC as a way to explore its possible use as a "surrogate" of tumor tissues to non-invasively assess the genomic landscape of the cancer and its evolution during treatment. Additionally, evidences confirm the existence of CTC in epithelial-to-mesenchymal transition (EMT) characterized by a variable loss of epithelial markers. Since the EMT process can originate cells with enhanced invasiveness, stemness and drug-resistance, the enumeration and characterization of this population, perhaps the one truly responsible of tumor recurrence and progression, could be more clinically useful. For these reasons, several devices able to capture CTC independently from the expression of epithelial markers have been developed. In this review, we will describe the types of heterogeneity so far identified and the key role played by the epithelial-to-mesenchymal transition in driving CTC heterogeneity. The clinical relevance of detecting CTC-heterogeneity will be discussed as well.


Asunto(s)
Neoplasias de la Mama/patología , Mama/patología , Células Neoplásicas Circulantes/patología , Animales , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/diagnóstico , Transición Epitelial-Mesenquimal , Femenino , Humanos , Metástasis de la Neoplasia/patología , Pronóstico
6.
Angew Chem Int Ed Engl ; 55(30): 8581-4, 2016 07 18.
Artículo en Inglés | MEDLINE | ID: mdl-27247024

RESUMEN

The number of circulating tumor cells (CTCs) in blood is strongly correlated with the progress of metastatic cancer. Current methods to detect CTCs are based on immunostaining or discrimination of physical properties. Herein, a label-free method is presented exploiting the abnormal metabolic behavior of cancer cells. A single-cell analysis technique is used to measure the secretion of acid from individual living tumor cells compartmentalized in microfluidically prepared, monodisperse, picoliter (pL) droplets. As few as 10 tumor cells can be detected in a background of 200 000 white blood cells and proof-of-concept data is shown on the detection of CTCs in the blood of metastatic patients.


Asunto(s)
Gotas Lipídicas/química , Microfluídica/métodos , Células Neoplásicas Circulantes/metabolismo , Benzopiranos/química , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Humanos , Leucocitos/citología , Leucocitos/metabolismo , Células Neoplásicas Circulantes/patología , Análisis de la Célula Individual , Espectrometría de Fluorescencia
7.
Breast Cancer Res ; 18(1): 30, 2016 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-26961140

RESUMEN

BACKGROUND: Although recent models suggest that the detection of Circulating Tumor Cells (CTC) in epithelial-to-mesenchymal transition (EM CTC) might be related to disease progression in metastatic breast cancer (MBC) patients, current detection methods are not efficient in identifying this subpopulation of cells. Furthermore, the possible association of EM CTC with both clinicopathological features and prognosis of MBC patients has still to be demonstrated. Aims of this study were: first, to optimize a DEPArray-based protocol meant to identify, quantify and sort single, viable EM CTC and, subsequently, to test the association of EM CTC frequency with clinical data. METHODS: This prospective observational study enrolled 56 MBC patients regardless of the line of treatment. Blood samples, depleted of CD45(pos) leukocytes, were stained with an antibody cocktail recognizing both epithelial and mesenchymal markers. Four CD45(neg) cell subpopulations were identified: cells expressing only epithelial markers (E CTC), cells co-expressing epithelial and mesenchymal markers (EM CTC), cells expressing only mesenchymal markers (MES) and cells negative for every tested marker (NEG). CTC subpopulations were quantified as both absolute cell count and relative frequency. The association of CTC subpopulations with clinicopathological features, progression free survival (PFS), and overall survival (OS) was explored by Wilcoxon-Mann-Whitney test and Univariate Cox Regression Analysis, respectively. RESULTS: By employing the DEPArray-based strategy, we were able to assess the presence of cells pertaining to the above-described classes in every MBC patient. We observed a significant association between specific CD45(neg) subpopulations and tumor subtypes (e.g. NEG and triple negative), proliferation (NEG and Ki67 expression) and sites of metastatic spread (e.g. E CTC and bone; NEG and brain). Importantly, the fraction of CD45(neg) cells co-expressing epithelial and mesenchymal markers (EM CTC) was significantly associated with poorer PFS and OS, computed, this latter, both from the diagnosis of a stage IV disease and from the initial CTC assessment. CONCLUSION: This study suggests the importance of dissecting the heterogeneity of CTC in MBC. Precise characterization of CTC could help in estimating both metastatization pattern and outcome, driving clinical decision-making and surveillance strategies.


Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias de la Mama/patología , Transición Epitelial-Mesenquimal/genética , Células Neoplásicas Circulantes , Pronóstico , Adulto , Neoplasias de la Mama/sangre , Neoplasias de la Mama/genética , Supervivencia sin Enfermedad , Femenino , Humanos , Persona de Mediana Edad , Metástasis de la Neoplasia
8.
Sci Rep ; 6: 21629, 2016 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-26899926

RESUMEN

The mesenchymal state in cancer is usually associated with poor prognosis due to the metastatic predisposition and the hyper-activated metabolism. Exploiting cell glucose metabolism we propose a new method to detect mesenchymal-like cancer cells. We demonstrate that the uptake of glucose-coated magnetic nanoparticles (MNPs) by mesenchymal-like cells remains constant when the glucose in the medium is increased from low (5.5 mM) to high (25 mM) concentration, while the MNPs uptake by epithelial-like cells is significantly reduced. These findings reveal that the glucose-shell of MNPs plays a major role in recognition of cells with high-metabolic activity. By selectively blocking the glucose transporter 1 channels we showed its involvement in the internalization process of glucose-coated MNPs. Our results suggest that glucose-coated MNPs can be used for metabolic-based assays aimed at detecting cancer cells and that can be used to selectively target cancer cells taking advantage, for instance, of the magnetic-thermotherapy.


Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Transportador de Glucosa de Tipo 1/genética , Glucosa/administración & dosificación , Nanopartículas de Magnetita/administración & dosificación , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Femenino , Glucosa/química , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/antagonistas & inhibidores , Humanos , Hipertermia Inducida , Células MCF-7 , Nanopartículas de Magnetita/química , Mesodermo/metabolismo , Mesodermo/patología
10.
Circulation ; 128(12): 1286-97, 2013 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-23983250

RESUMEN

BACKGROUND: Little is known about the function of inositol 1,4,5-trisphosphate receptors (IP3Rs) in the adult heart experimentally. Moreover, whether these Ca(2+) release channels are present and play a critical role in human cardiomyocytes remains to be defined. IP3Rs may be activated after Gαq-protein-coupled receptor stimulation, affecting Ca(2+) cycling, enhancing myocyte performance, and potentially favoring an increase in the incidence of arrhythmias. METHODS AND RESULTS: IP3R function was determined in human left ventricular myocytes, and this analysis was integrated with assays in mouse myocytes to identify the mechanisms by which IP3Rs influence the electric and mechanical properties of the myocardium. We report that IP3Rs are expressed and operative in human left ventricular myocytes. After Gαq-protein-coupled receptor activation, Ca(2+) mobilized from the sarcoplasmic reticulum via IP3Rs contributes to the decrease in resting membrane potential, prolongation of the action potential, and occurrence of early afterdepolarizations. Ca(2+) transient amplitude and cell shortening are enhanced, and extrasystolic and dysregulated Ca(2+) elevations and contractions become apparent. These alterations in the electromechanical behavior of human cardiomyocytes are coupled with increased isometric twitch of the myocardium and arrhythmic events, suggesting that Gαq-protein-coupled receptor activation provides inotropic reserve, which is hampered by electric instability and contractile abnormalities. Additionally, our findings support the notion that increases in Ca(2+) load by IP3Rs promote Ca(2+) extrusion by forward-mode Na(+)/Ca(2+) exchange, an important mechanism of arrhythmic events. CONCLUSIONS: The Gαq-protein/coupled receptor/IP3R axis modulates the electromechanical properties of the human myocardium and its propensity to develop arrhythmias.


Asunto(s)
Potenciales de Acción/fisiología , Señalización del Calcio/fisiología , Insuficiencia Cardíaca/fisiopatología , Receptores de Inositol 1,4,5-Trifosfato/fisiología , Miocitos Cardíacos/fisiología , Adulto , Animales , Arritmias Cardíacas/fisiopatología , Células Cultivadas , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/fisiología , Insuficiencia Cardíaca/genética , Ventrículos Cardíacos/citología , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Contracción Miocárdica/fisiología , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Retículo Sarcoplasmático/fisiología , Transducción de Señal/fisiología
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