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In the early stages of metastasis, cancer cells exit the primary tumor and enter the vasculature. Although most studies have focused on the tumor invasive front, cancer cells from the tumor core can also potentially metastasize. To address cell motility in the tumor core, we imaged tumor explants from spontaneously forming tumors in mice in real time using long-term two-photon microscopy. Cancer cells in the tumor core are remarkably dynamic and exhibit correlated migration patterns, giving rise to local 'currents' and large-scale tissue dynamics. Although cells exhibit stop-and-start migration with intermittent pauses, pausing does not appear to be required during division. Use of pharmacological inhibitors indicates that migration patterns in tumors are actively driven by the actin cytoskeleton. Under these conditions, we also observed a relationship between migration speed and correlation length, suggesting that cells in tumors are near a jamming transition. Our study provides new insight into the dynamics of cancer cells in the tumor core, opening new avenues of research in understanding the migratory properties of cancer cells and later metastasis.This article has an associated First Person interview with the first author of the paper.
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Citoesqueleto de Actina/patología , Movimiento Celular , Células Neoplásicas Circulantes/patología , Animales , Carcinogénesis/inducido químicamente , Ratones , Microscopía de Fluorescencia por Excitación Multifotónica , Neoplasias Experimentales , Cultivo Primario de Células , Tamoxifeno/farmacologíaRESUMEN
Anal squamous cell carcinoma (ASCC) is a rare tumour, but its incidence is increasing. Standard chemoradiotherapy fails to cure 20% of patients and no targeted therapy is currently approved for recurrent ASCC. The PI3K/Akt/mTOR pathway is frequently altered in this poorly characterised carcinoma. IGF2 was identified here as a key factor in ASCC oncogenesis, as IGF2 was shown to play a crucial role in the PI3K pathway with frequent (~60%) and mutually exclusive genomic alterations in IGF2, IGF1R, PTEN and PIK3CA genes. We also demonstrated that IGF2 expression is mainly due to cancer-associated fibroblasts and that IGF2 secreted by cancer-associated fibroblasts from ASCC samples promotes proliferation of a human ASCC cell line via IGF2 paracrine signalling. Altogether, these results highlight the key role of the IGF2/PI3K axis, and the major role of cancer-associated fibroblasts in tumour growth via IGF2 secretion, suggesting a major role of IGF2/IGF1R inhibitors in ASCC therapies.
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Neoplasias del Ano/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma de Células Escamosas/metabolismo , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/metabolismo , Animales , Neoplasias del Ano/genética , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Mutación , Trasplante de Neoplasias , Comunicación Paracrina , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: A better understanding of the molecular profile of anal squamous cell carcinomas (ASCCs) is necessary to consider new therapeutic approaches, and the identification of prognostic and predictive factors for response to treatment. METHODS: We retrospectively analysed tumours from ASCC patients for mutational analysis of KRAS, NRAS, HRAS, BRAF, PIK3CA, MET, TP53 and FBXW7 genes by HRM and Sanger sequencing analysis. RESULTS: Specimens from 148 patients were analysed: 96 treatment-naive tumours and 52 recurrences after initial radiotherapy (RT) or chemoradiotherapy (CRT). Mutations of KRAS, PIK3CA, FBXW7 and TP53 genes were present in 3 (2.0%), 30 (20.3%), 9 (6.1%) and 7 tumours (4.7%), respectively. The distribution of the mutations was similar between treatment-naive tumours and recurrences, except for TP53 mutations being more frequent in recurrences (P=0.0005). In patients treated with abdominoperineal resection (APR) after relapse (n=38, median follow-up of 18.2 years), overall survival (OS) was significantly correlated with HPV16 status (P=0.048), gender (P=0.045) and PIK3CA mutation (P=0.037). The PIK3CA status retained its prognostic significance in Cox multivariate regression analysis (P=0.025). CONCLUSIONS: Our study identified PIK3CA mutation as an independent prognostic factor in patients who underwent APR for ASCC recurrence, suggesting a potential benefit from adjuvant treatment and the evaluation of targeted therapies with PI3K/Akt/mTor inhibitors in PIK3CA-mutated patients.
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Neoplasias del Ano/genética , Neoplasias del Ano/cirugía , Mutación , Fosfatidilinositol 3-Quinasas/genética , Terapia Recuperativa/métodos , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias del Ano/metabolismo , Neoplasias del Ano/patología , Fosfatidilinositol 3-Quinasa Clase I , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/cirugía , Fosfatidilinositol 3-Quinasas/metabolismo , Estudios RetrospectivosRESUMEN
BACKGROUND: The B-subunit of Shiga toxin (STxB) specifically binds to the glycosphingolipid Gb3 that is highly expressed on a number of human tumors and has been shown to target tumor cells in mouse models and ex vivo on primary colon carcinoma specimen. METHODS: Using a novel ex vivo STxB labeling (ESL) method we studied Gb3 expression in cytological specimens of primary human breast tumors from 107 patients, and in synchronous lymph node metastases from 20 patients. Fluorescent STxB was incubated with fine-needle aspiration (FNA) specimens, and Gb3 expression was evaluated by fluorescence microscopy. Furthermore, 11 patient-derived human breast cancer xenografts (HBCx) were evaluated for expression of Gb3 by ESL and FACS. In addition, the biodistribution of fluorescent STxB conjugate was studied after intravenous injection in a Gb3 positive HBCx model. RESULTS: Gb3 expression was detected in 62 of 107 patients (57.9%), mainly in epithelial tumor cells. Gb3 positivity correlated with estrogen receptor expression (p≤0.01), whereas absence of Gb3 expression in primary tumors was correlated with the presence of lymph node metastases (p≤0.03). 65% of lymph node metastases were Gb3 positive and in 40% of tested patients, we observed a statistically significant increase of metastatic Gb3 expression (p≤0.04). Using concordant ESL and flow cytometry analysis, 6 out of 11 HBCx samples were scored positive. Intravenous injections of fluorescent STxB into HBC xenografted mice showed preferential STxB accumulation in epithelial cells and cells with endothelial morphology of the tumor. CONCLUSION: The enhanced expression of Gb3 in primary breast carcinomas and its lymph node metastases indicate that the development of STxB-based therapeutic strategies is of interest in this pathology. Gb3 expressing HBCx can be used as a model for preclinical studies with STxB conjugates. Finally, the ESL technique on FNA represents a rapid and cost effective method for the stratification of patients in future clinical trials.
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Adenofibroma/química , Antígenos de Carbohidratos Asociados a Tumores/análisis , Neoplasias de la Mama/química , Carcinoma/química , Toxinas Shiga/farmacocinética , Animales , Biopsia con Aguja Fina , Mama/química , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma/metabolismo , Carcinoma/secundario , Sistemas de Liberación de Medicamentos , Femenino , Citometría de Flujo , Humanos , Inyecciones Intravenosas , Metástasis Linfática , Glándulas Mamarias Humanas/química , Ratones , Microscopía Fluorescente , Persona de Mediana Edad , Receptores de Estrógenos/análisis , Toxinas Shiga/administración & dosificaciónRESUMEN
BACKGROUND: Tumor endothelial transdifferentiation and VEGFR1/2 expression by cancer cells have been reported in glioblastoma but remain poorly documented for many other cancer types. METHODS: To characterize vasculature of patient-derived tumor xenografts (PDXs), largely used in preclinical anti-angiogenic assays, we designed here species-specific real-time quantitative RT-PCR assays. Human and mouse PECAM1/CD31, ENG/CD105, FLT1/VEGFR1, KDR/VEGFR2 and VEGFA transcripts were analyzed in a large series of 150 PDXs established from 8 different tumor types (53 colorectal, 14 ovarian, 39 breast and 15 renal cell cancers, 6 small cell and 5 non small cell lung carcinomas, 13 cutaneous melanomas and 5 glioblastomas) and in two bevacizumab-treated non small cell lung carcinomas xenografts. RESULTS: As expected, mouse cell proportion in PDXs -evaluated by quantifying expression of the housekeeping gene TBP- correlated with all mouse endothelial markers and human VEGFA RNA levels. More interestingly, we observed human PECAM1/CD31 and ENG/CD105 expression in all tumor types, with higher rate in glioblastoma and renal cancer xenografts. Human VEGFR expression profile varied widely depending on tumor types with particularly high levels of human FLT1/VEGFR1 transcripts in colon cancers and non small cell lung carcinomas, and upper levels of human KDR/VEGFR2 transcripts in non small cell lung carcinomas. Bevacizumab treatment induced significant low expression of mouse Pecam1/Cd31, Eng/Cd105, Flt1/Vegfr1 and Kdr/Vefr2 while the human PECAM1/CD31 and VEGFA were upregulated. CONCLUSIONS: Taken together, our results strongly suggest existence of human tumor endothelial cells in all tumor types tested and of both stromal and tumoral autocrine VEGFA-VEGFR1/2 signalings. These findings should be considered when evaluating molecular mechanisms of preclinical response and resistance to tumor anti-angiogenic strategies.
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Células Endoteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Neoplasias Experimentales/genética , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/genética , Inhibidores de la Angiogénesis/farmacología , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Bevacizumab , Biomarcadores de Tumor/metabolismo , Células Endoteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVES: To evaluate radiographically the quality of root canal fillings and compare manual and rotary preparation performed on extracted teeth by undergraduate dental students. STUDY DESIGN: A total of 561 premolars and molars extracted teeth were prepared using nickel-titanium rotary files or manual instrumentation and filled with gutta-percha using a cold lateral condensation technique, by 4th grade undergraduate students. Periapical radiographs were used to assess the technical quality of the root canal filling, evaluating three variables: length, density and taper. These data were recorded, scored and used to study the "technical success rate" and the "overall score". The length of each root canal filling was classified as acceptable, short and overfilled, based on their relationship with the radiographic apex. Density and taper of filling were evaluated based on the presence of voids and the uniform tapering of the filling, respectively. Statistical analysis was used to evaluate the quality of root canal treatment, considering p < 0.05 as a statistical significant level. RESULTS: The percentage of technical success was 44% and the overall score was 7.8 out of 10. Technical success and overall score were greater with rotary instruments (52% against 28% with a manual one, p < 0.001; 8.3 against 6.7 respectively, p < 0.001). CONCLUSIONS: It appears that inexperienced operators perform better root canal treatment (RCT) with the use of rotary instrumentation.
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Competencia Clínica , Endodoncia/educación , Tratamiento del Conducto Radicular/normas , Diente/diagnóstico por imagen , Humanos , Técnicas In Vitro , Radiografía , Facultades de Odontología , España , Estudiantes de OdontologíaRESUMEN
Intestinal inflammation shifts microbiota composition and metabolism. How the host monitors and responds to such changes remains unclear. Here, we describe a protective mechanism by which mucosal-associated invariant T (MAIT) cells detect microbiota metabolites produced upon intestinal inflammation and promote tissue repair. At steady state, MAIT ligands derived from the riboflavin biosynthesis pathway were produced by aerotolerant bacteria residing in the colonic mucosa. Experimental colitis triggered luminal expansion of riboflavin-producing bacteria, leading to increased production of MAIT ligands. Modulation of intestinal oxygen levels suggested a role for oxygen in inducing MAIT ligand production. MAIT ligands produced in the colon rapidly crossed the intestinal barrier and activated MAIT cells, which expressed tissue-repair genes and produced barrier-promoting mediators during colitis. Mice lacking MAIT cells were more susceptible to colitis and colitis-driven colorectal cancer. Thus, MAIT cells are sensitive to a bacterial metabolic pathway indicative of intestinal inflammation.
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Colitis , Disbiosis , Microbioma Gastrointestinal , Ratones Endogámicos C57BL , Células T Invariantes Asociadas a Mucosa , Animales , Células T Invariantes Asociadas a Mucosa/inmunología , Colitis/inmunología , Colitis/microbiología , Disbiosis/inmunología , Ratones , Microbioma Gastrointestinal/inmunología , Ratones Noqueados , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Riboflavina/inmunologíaRESUMEN
The microenvironment plays an essential role in tumor development and metastatic progression. Here, we describe a simple and rapid protocol to generate tumors in mice using colon cancer cell lines or tumoroids in the correct microenvironment, colonic mucosa. We also detail steps for monitoring the growth of the primary tumor in real time using colonoscopy or in vivo imaging system, as well as monitoring metastasis development. Finally, we describe tissue collection and sample preparation for subsequent immunohistochemistry analysis.
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Neoplasias del Colon , Ratones , Animales , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Inmunohistoquímica , Microambiente TumoralRESUMEN
During tumor progression, cancer-associated fibroblasts (CAFs) accumulate in tumors and produce an excessive extracellular matrix (ECM), forming a capsule that enwraps cancer cells. This capsule acts as a barrier that restricts tumor growth leading to the buildup of intratumoral pressure. Combining genetic and physical manipulations in vivo with microfabrication and force measurements in vitro, we found that the CAFs capsule is not a passive barrier but instead actively compresses cancer cells using actomyosin contractility. Abrogation of CAFs contractility in vivo leads to the dissipation of compressive forces and impairment of capsule formation. By mapping CAF force patterns in 3D, we show that compression is a CAF-intrinsic property independent of cancer cell growth. Supracellular coordination of CAFs is achieved through fibronectin cables that serve as scaffolds allowing force transmission. Cancer cells mechanosense CAF compression, resulting in an altered localization of the transcriptional regulator YAP and a decrease in proliferation. Our study unveils that the contractile capsule actively compresses cancer cells, modulates their mechanical signaling, and reorganizes tumor morphology.
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Fibroblastos Asociados al Cáncer , Neoplasias , Fibroblastos Asociados al Cáncer/patología , Mecanotransducción Celular , Línea Celular Tumoral , Fibroblastos/patología , Microambiente Tumoral , Neoplasias/patologíaRESUMEN
Tumours are complex ecosystems composed of different types of cells that communicate and influence each other. While the critical role of stromal cells in affecting tumour growth is well established, the impact of mutant cancer cells on healthy surrounding tissues remains poorly defined. Here, using mouse intestinal organoids, we uncover a paracrine mechanism by which intestinal cancer cells reactivate foetal and regenerative YAP-associated transcriptional programmes in neighbouring wildtype epithelial cells, rendering them adapted to thrive in the tumour context. We identify the glycoprotein thrombospondin-1 (THBS1) as the essential factor that mediates non-cell-autonomous morphological and transcriptional responses. Importantly, Thbs1 is associated with bad prognosis in several human cancers. This study reveals the THBS1-YAP axis as the mechanistic link mediating paracrine interactions between epithelial cells in intestinal tumours.
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Proteínas Adaptadoras Transductoras de Señales , Neoplasias , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Ecosistema , Células Epiteliales/metabolismo , Ratones , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
SF3B1 mutations are recurrent in cancer and result in aberrant splicing of a previously defined set of genes. Here, we investigated the fate of aberrant transcripts induced by mutant SF3B1 and the related functional consequences. We first demonstrate that mutant SF3B1 does not alter global nascent protein synthesis, suggesting target-dependent consequences. Polysome profiling revealed that 35% of aberrantly spliced transcripts are more translated than their corresponding canonically spliced transcripts. This mostly occurs in genes with enriched metabolic functions. Furthermore, LC-MS/MS analysis showed that mutant SF3B1 impacts the abundance of proteins involved in metabolism. Functional metabolic characterization revealed that mutant SF3B1 decreases mitochondrial respiration and promotes glycolysis to compensate for defective mitochondrial metabolism. Hence, mutant SF3B1 induces glycolysis dependency, which sensitizes cells to glycolysis inhibition. Overall, we provide evidence of the oncogenic involvement of mutant SF3B1 in uveal melanoma through a metabolic switch to glycolysis, revealing vulnerability to glycolysis inhibitors as a promising therapeutic strategy.
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Patient-derived tumor xenograft (PDX) is now largely recognized as a key preclinical model for cancer research, mimicking patient tumor phenotype and genotype. Immunodeficient mice, well-known to develop spontaneous lymphoma, are required for PDX growth. As for all animal models used for further clinical translation, a robust experimental design is strongly required to lead to conclusive results. Here we briefly report unintentional co-engraftment of mouse lymphoma during expansion of well-established PDXs to illustrate the importance of systematic check of the PDX identity to avoid misinterpretation. Besides, this quality control based on complementary approaches deserves a more detailed description in materials and methods section to ensure experimental validity and reproducibility.
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BACKGROUND: This study aimed to evaluate the area of dentin growth in rabbit incisors after pulp capping with plasma rich in growth factors (PRGF) compared with mineral trioxide aggregate (MTA) by fluorescence. METHODS: twenty-seven upper and lower incisors of rabbits were divided into 4 groups: poor PRGF (F1) (n = 9 teeth), rich PRGF (F2) (n = 8 teeth), ProRoot MTA (positive control, n = 5 teeth), and untreated (NC) (negative control, n = 5). Fluorochrome markers were injected 24 h before surgery and the day before euthanasia, 28 days after the vital pulp therapy (VPT). Two transverse cuts were performed to every tooth: the first cut (A), 1 mm incisal to the gingival margin, and the second cut (B), 5 mm apical to the first cut. The sections were assessed with histomorphometric evaluation by fluorescence microscopy, comparing the dentin area between fluorescence marks and the total mineralized area. RESULTS: The higher percentage of dentin growth was observed in the F2 group (B = 63.25%, A = 36.52%), followed by F1 (B = 57.63%, A = 30,12%) and MTA (B = 38.64%, A = 15.74%). The group with lowest percentage of dentin growth was the NC group (B = 29.22%, A = 7.82%). Significant difference (p < 0.05) was found between F2 group and MTA, also statistically significant difference has been observed comparing dentin growth areas of NC group with F1 and F2 groups. CONCLUSIONS: The application of PRGF rich and poor fraction as a pulp capping material stimulated dentin formation more intensively than MTA and NC.
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BACKGROUND: Multicellular tumour sphere models have been shown to closely mimic phenotype characteristics of in vivo solid tumours, or to allow in vitro propagation of cancer stem cells (CSCs). CSCs are usually characterized by the expression of specific membrane markers using flow cytometry (FC) after enzymatic dissociation. Consequently, the spatial location of positive cells within spheres is not documented. Confocal microscopy is the best technique for the imaging of thick biological specimens after multi-labelling but suffers from poor antibody penetration. Thus, we describe here a new protocol for in situ confocal imaging of protein expression in intact spheroids. METHODS: Protein expression in whole spheroids (150 mum in diameter) from two human colon cancer cell lines, HT29 and CT320X6, has been investigated with confocal immunostaining, then compared with profiles obtained through paraffin immunohistochemistry (pIHC) and FC. Target antigens, relevant for colon cancer and with different expression patterns, have been studied. RESULTS: We first demonstrate that our procedure overcomes the well-known problem of antibody penetration in compact structures by performing immunostaining of EpCAM, a membrane protein expressed by all cells within our spheroids. EpCAM expression is detected in all cells, even the deepest ones. Likewise, antibody access is confirmed with CK20 and CD44 immunostaining. Confocal imaging shows that 100% of cells express beta-catenin, mainly present in the plasma membrane with also cytoplasmic and nuclear staining, in agreement with FC and pIHC data. pIHC and confocal imaging show similar CA 19-9 cytoplasmic and membranar expression profile in a cell subpopulation. CA 19-9+ cell count confirms confocal imaging as a highly sensitive method (75%, 62% and 51%, for FC, confocal imaging and pIHC, respectively). Finally, confocal imaging reveals that the weak expression of CD133, a putative colon CSC marker, is restricted to the luminal cell surface of colorectal cancer acini, with CD133+ cellular debris into glandular lumina. CONCLUSION: The present protocol enables in situ visualization of protein expression in compact three-dimensional models by whole mount confocal imaging, allowing the accurate localization and quantification of cells expressing specific markers. It should prove useful to study rare events like CSCs within tumour spheres.
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Regulación Neoplásica de la Expresión Génica , Microscopía Confocal/métodos , Neoplasias/patología , Células Madre Neoplásicas/citología , Antígeno AC133 , Antígenos CD/biosíntesis , Línea Celular Tumoral , Citometría de Flujo/métodos , Perfilación de la Expresión Génica , Glicoproteínas/biosíntesis , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Inmunohistoquímica/métodos , PéptidosRESUMEN
Obtaining representative human colon cancer cell lines from fresh tumors is technically difficult. Using 32 tumor fragments from patients with colon cancer, the present study shows that prior xenograft leads to more efficient cell line establishment compared with direct establishment from fresh tumors (P < 0.05). From 26 tumor specimens, we successfully established 20 tumor xenografts in nude mice (77%); among 19 of these xenografts, 9 (47%) led to cell lines, including four from liver metastases. Only 3 of 31 tumor specimens (9.7%) grew immediately in vitro, and all were derived from primary tumors. To compare major phenotypic and genotypic characteristics of human colon cancer cell lines derived from the same tumor fragment using two protocols, the two pairs of cell lines obtained from 2 of 32 tumor fragments were extensively studied. They displayed similar morphology and were able to form compact spheroids. Chemosensitivity to 5-fluorouracil, CPT11, and L-OHP differed between cell lines obtained from patient tumors and those derived from xenografts. Matched cell lines shared a common core of karyotype alterations and distinctive additional chromosomal aberrations. Expression levels of genes selected for their role in oncogenesis evaluated by real-time quantitative PCR were found to be statistically correlated whatever the in vitro culture model used. In conclusion, xenotransplantation in mice of tumor fragments before establishment of cell lines enables generation of more novel human cancer cell lines for investigation of colon cancer cell biology, opening up the opportunity of reproducing the diversity of this disease.
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Línea Celular Tumoral , Neoplasias del Colon/patología , Animales , Procesos de Crecimiento Celular/fisiología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Ensayos de Selección de Medicamentos Antitumorales , Perfilación de la Expresión Génica , Humanos , Cariotipificación , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante HeterólogoRESUMEN
Steady-state turnover is a hallmark of epithelial tissues throughout adult life. Intestinal epithelial turnover is marked by continuous cell migration, which is assumed to be driven by mitotic pressure from the crypts. However, the balance of forces in renewal remains ill-defined. Combining biophysical modeling and quantitative three-dimensional tissue imaging with genetic and physical manipulations, we revealed the existence of an actin-related protein 2/3 complex-dependent active migratory force, which explains quantitatively the profiles of cell speed, density, and tissue tension along the villi. Cells migrate collectively with minimal rearrangements while displaying dual-apicobasal and front-back-polarity characterized by actin-rich basal protrusions oriented in the direction of migration. We propose that active migration is a critical component of gut epithelial turnover.
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Movimiento Celular/fisiología , Mucosa Intestinal/citología , Mucosa Intestinal/fisiología , Mitosis , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/fisiología , Animales , Movimiento Celular/genética , Polaridad Celular , Imagenología Tridimensional , Mucosa Intestinal/metabolismo , Ratones Noqueados , Modelos BiológicosRESUMEN
Genomic alterations of anal squamous cell carcinoma (ASCC) remain poorly understood due to the rarity of this tumor. Array comparative genomic hybridization and targeted gene sequencing were performed in 49 cases of ASCC. The most frequently altered regions (with a frequency greater than 25%) were 10 deleted regions (2q35, 2q36.3, 3p21.2, 4p16.3, 4p31.21, 7q36.1, 8p23.3, 10q23.2, 11q22.3, and 13q14.11) and 8 gained regions (1p36.33, 1q21.1, 3q26.32, 5p15.33, 8q24.3, 9q34.3, 16p13.3, and 19p13.3). The most frequent minimal regions of deletion (55%) encompassed the 11q22.3 region containing ATM, while the most frequent minimal regions of gain (57%) encompassed the 3q26.32 region containing PIK3CA. Recurrent homozygous deletions were observed for 5 loci (ie, TGFR2 in 4 cases), and recurrent focal amplifications were observed for 8 loci (ie, DDR2 and CCND1 in 3 cases, respectively). Several of the focal amplified genes are targets for specific therapies. Integrated analysis showed that the PI3K/Akt/mTOR signaling pathway was the pathway most extensively affected, particularly in recurrences compared to treatment-naive tumors (64% vs 30%; P = .017). In patients with ASCC recurrences, poor overall survival (OS) was significantly correlated with a large number of altered regions (P = .024). These findings provide insight into the somatic genomic alterations in ASCC and highlight the key role of the druggable PI3K/Akt/mTOR signaling pathway.
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Anal squamous cell carcinomas (ASCC) are rare tumours in humans. The etiological role of HPV infection is now well established but little is known about the molecular landscape and signalling pathways involved in the pathogenesis of this cancer. Here we report the results from a whole exome sequencing of a homogeneous group of 20 treatment-naive ASCC. A total of 2422 somatic single nucleotide variations (SNV) were found, with an overall moderate rate of somatic mutations per tumour (median: 105 relevant SNV per tumour) but a high mutational load in 3 tumours. The mutational signatures associated with age and APOBEC were observed in 100% and 60% of tumours respectively. The most frequently mutated genes were PIK3CA (25%) followed by FBXW7 (15%), FAT1 (15%), and TRIP12 (15%), the two last ones having never been described in ASCC. The main copy number alterations were gains of chromosome 3q (affecting PIK3CA) and losses of chromosome 11q (affecting ATM). The combined analysis of somatic mutations and copy number alterations show that recurrent alterations of the PI3K/AKT/mTOR pathway are frequent (60%) in these tumours, as well as potentially targetable alterations of other signalling pathways that have never been described in ASCC such as chromatin remodelling (45%) and ubiquitin mediated proteolysis (35%). These results highlight the possible implication of these aberrant signalling pathways in anal carcinogenesis and suggest promising new therapeutic approaches in ASCC. The high somatic mutation burden found in some tumours, suggesting an elevated neoantigen load could also predict sensitivity of ASCC to immunotherapy.
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OBJECTIVE: The objective of the study was to evaluate maternal serum pro-early placenta insulin like (proEPIL) levels during normal and pathologic pregnancy by using a newly developed enzyme-linked immunosorbent assay, based on a monoclonal antibody designated EPIL15 and directed to the pro-EPIL C-chain 98-108 region. STUDY DESIGN: In a group of healthy pregnant women (n = 22), proEPIL peptide serum levels were measured longitudinally throughout gestation (8-12, 20-24, and 30-34 weeks). Serum proEPIL levels were measured in women with preterm labor (n = 24), intrauterine growth restriction (n = 27), and preeclampsia (n = 12). RESULTS: In healthy pregnant women, a significant rise of serum pro-EPIL levels (mean +/- SEM) was observed during the third trimester of gestation (30.97 +/- 2.978 ng/mL; P < .01), with the highest serum levels at 30-34 weeks' gestation (P < .001). Serum proEPIL levels were found elevated in women with intrauterine growth restriction (107.4 +/- 12.99 ng/mL), preeclampsia (104.8 +/- 36.20 ng/mL), or preterm labor (183.8 +/- 36.42 ng/mL) in comparison with levels observed in healthy pregnant women (P < .001). CONCLUSION: These results showed that proEPIL secretion increases in the last trimester during normal pregnancy and is highly secreted in women with pathologic conditions.
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Retardo del Crecimiento Fetal/sangre , Péptidos y Proteínas de Señalización Intercelular/sangre , Trabajo de Parto Prematuro/sangre , Preeclampsia/sangre , Embarazo/sangre , Adulto , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Tercer Trimestre del Embarazo/sangreRESUMEN
The placenta relies on phenotypes that are characteristic of cancer to successfully implant the embryo in the uterus during early pregnancy. Notably, it has to invade its host tissues, promote angiogenesis-while surviving hypoxia-, and escape the immune system. Similarities in DNA methylation patterns between the placenta and cancers suggest that common epigenetic mechanisms may be involved in regulating these behaviors. We show here that megabase-scale patterns of hypomethylation distinguish first from third trimester chorionic villi in the placenta, and that these patterns mirror those that distinguish many tumors from corresponding normal tissues. We confirmed these findings in villous cytotrophoblasts isolated from the placenta and identified a time window at the end of the first trimester, when these cells come into contact with maternal blood, as the likely time period for the methylome alterations. Furthermore, the large genomic regions affected by these patterns of hypomethylation encompass genes involved in pathways related to epithelial-mesenchymal transition, immune response, and inflammation. Analyses of expression profiles corresponding to genes in these hypomethylated regions in colon adenocarcinoma tumors point to networks of differentially expressed genes previously implicated in carcinogenesis and placentogenesis, where nuclear factor kappa B is a key hub. Taken together, our results suggest the existence of epigenetic switches involving large-scale changes of methylation in the placenta during pregnancy and in tumors during neoplastic transformation. The characterization of such epigenetic switches might lead to the identification of biomarkers and drug targets in oncology as well as in obstetrics and gynecology.