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The metastatic progression of cancer remains a major issue in patient treatment. However, the molecular and cellular mechanisms underlying this process remain unclear. Here, we use primary explants and organoids from patients harboring mucinous colorectal carcinoma (MUC CRC), a poor-prognosis histological form of digestive cancer, to study the architecture, invasive behavior and chemoresistance of tumor cell intermediates. We report that these tumors maintain a robust apico-basolateral polarity as they spread in the peritumoral stroma or organotypic collagen-I gels. We identified two distinct topologies - MUC CRCs either display a conventional 'apical-in' polarity or, more frequently, harbor an inverted 'apical-out' topology. Transcriptomic analyses combined with interference experiments on organoids showed that TGFß and focal adhesion signaling pathways are the main drivers of polarity orientation. Finally, we show that the apical-out topology is associated with increased resistance to chemotherapeutic treatments in organoids and decreased patient survival in the clinic. Thus, studies on patient-derived organoids have the potential to bridge histological, cellular and molecular analyses to decrypt onco-morphogenic programs and stratify cancer patients. This article has an associated First Person interview with the first author of the paper.
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Neoplasias Colorrectales , Organoides , Adhesión Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Humanos , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Coping ethically with dramatic changes such as those occurring in times of pandemics is a difficult challenge for animal facilities and for researchers using animals for scientific purposes. Managing such situations is impossible without a specific contingency plan. However, because pandemics are rare events, they have not been included in some disaster plans. We present here various ways to manage the broad and rapid changes that may be necessary during a pandemic, focusing on actions for optimizing the conservation of animals while ensuring continuous high standards of animal welfare. The proposed approach is graduated and encompasses research, researchers, animal caretakers, supply chains, and logistics.
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Planificación en Desastres , Gripe Humana , Animales , Humanos , Gripe Humana/epidemiología , Pandemias/prevención & controlRESUMEN
ß-thalassemia major (ß-TM) is an inherited hemoglobinopathy caused by a quantitative defect in the synthesis of ß-globin chains of hemoglobin, leading to the accumulation of free a-globin chains that aggregate and cause ineffective erythropoiesis. We have previously demonstrated that terminal erythroid maturation requires a transient activation of caspase-3 and that the chaperone Heat Shock Protein 70 (HSP70) accumulates in the nucleus to protect GATA-1 transcription factor from caspase-3 cleavage. This nuclear accumulation of HSP70 is inhibited in human ß-TM erythroblasts due to HSP70 sequestration in the cytoplasm by free a-globin chains, resulting in maturation arrest and apoptosis. Likewise, terminal maturation can be restored by transduction of a nuclear-targeted HSP70 mutant. Here we demonstrate that in normal erythroid progenitors, HSP70 localization is regulated by the exportin-1 (XPO1), and that treatment of ß-thalassemic erythroblasts with an XPO1 inhibitor increased the amount of nuclear HSP70, rescued GATA-1 expression and improved terminal differentiation, thus representing a new therapeutic option to ameliorate ineffective erythropoiesis of ß-TM.
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Carioferinas , Receptores Citoplasmáticos y Nucleares , Talasemia beta , Diferenciación Celular , Eritroblastos , Eritropoyesis , Humanos , Carioferinas/genética , Receptores Citoplasmáticos y Nucleares/genética , Talasemia beta/tratamiento farmacológico , Talasemia beta/genética , Proteína Exportina 1RESUMEN
Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). CALR mutations, including the two most frequent 52 bp deletion (del52) and 5 bp insertion (ins5), induce a frameshift to the same alternative reading frame generating new C-terminal tails. In patients, del52 and ins5 induce two phenotypically distinct myeloproliferative neoplasms (MPNs). They are equally found in ET, but del52 is more frequent in PMF. We generated heterozygous and homozygous conditional inducible knock-in (KI) mice expressing a chimeric murine CALR del52 or ins5 with the human mutated C-terminal tail to investigate their pathogenic effects on hematopoiesis. Del52 induces greater phenotypic changes than ins5 including thrombocytosis, leukocytosis, splenomegaly, bone marrow hypocellularity, megakaryocytic lineage amplification, expansion and competitive advantage of the hematopoietic stem cell compartment. Homozygosity amplifies these features, suggesting a distinct contribution of homozygous clones to human MPNs. Moreover, homozygous del52 KI mice display features of a penetrant myelofibrosis-like disorder with extramedullary hematopoiesis linked to splenomegaly, megakaryocyte hyperplasia and the presence of reticulin fibers. Overall, modeling del52 and ins5 mutations in mice successfully recapitulates the differences in phenotypes observed in patients.
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Calreticulina/genética , Mielofibrosis Primaria/genética , Trombocitemia Esencial/genética , Animales , Calreticulina/metabolismo , Modelos Animales de Enfermedad , Femenino , Células Madre Hematopoyéticas/metabolismo , Homocigoto , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mutagénesis Insercional , Fenotipo , Mielofibrosis Primaria/metabolismo , Eliminación de Secuencia , Trombocitemia Esencial/metabolismoRESUMEN
Bone marrow (BM) niche cells help to keep adult hematopoietic stem cells (HSCs) in a quiescent state via secreted factors and induction of cell-cycle inhibitors. Here, we demonstrate that the adapter protein CABLES1 is a key regulator of long-term hematopoietic homeostasis during stress and aging. Young mice lacking Cables1 displayed hyperproliferation of hematopoietic progenitor cells. This defect was cell intrinsic, since it was reproduced in BM transplantation assays using wild-type animals as recipients. Overexpression and short hairpin RNA-mediated depletion of CABLES1 protein resulted in p21Cip/waf up- and downregulation, respectively. Aged mice lacking Cables1 displayed abnormalities in peripheral blood cell counts accompanied by a significant reduction in HSC compartment, concomitant with an increased mobilization of progenitor cells. In addition, Cables1-/- mice displayed increased sensitivity to the chemotherapeutic agent 5-fluorouracil due to an abnormal microenvironment. Altogether, our findings uncover a key role for CABLES1 in HSC homeostasis and stress hematopoiesis.
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Ciclinas/genética , Células Madre Hematopoyéticas/citología , Envejecimiento , Animales , Antígenos CD34/metabolismo , Células de la Médula Ósea/citología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclinas/antagonistas & inhibidores , Ciclinas/metabolismo , Fluorouracilo/farmacología , Hematopoyesis/efectos de los fármacos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Humanos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
Metastases account for 90% of cancer-related deaths; thus, it is vital to understand the biology of tumour dissemination. Here, we collected and monitored >50 patient specimens ex vivo to investigate the cell biology of colorectal cancer (CRC) metastatic spread to the peritoneum. This reveals an unpredicted mode of dissemination. Large clusters of cancer epithelial cells displaying a robust outward apical pole, which we termed tumour spheres with inverted polarity (TSIPs), were observed throughout the process of dissemination. TSIPs form and propagate through the collective apical budding of hypermethylated CRCs downstream of canonical and non-canonical transforming growth factor-ß signalling. TSIPs maintain their apical-out topology and use actomyosin contractility to collectively invade three-dimensional extracellular matrices. TSIPs invade paired patient peritoneum explants, initiate metastases in mice xenograft models and correlate with adverse patient prognosis. Thus, despite their epithelial architecture and inverted topology TSIPs seem to drive the metastatic spread of hypermethylated CRCs.
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Biomarcadores de Tumor/genética , Movimiento Celular , Polaridad Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Metilación de ADN , Células Epiteliales/patología , Neoplasias Peritoneales/genética , Neoplasias Peritoneales/secundario , Animales , Biomarcadores de Tumor/metabolismo , Células CACO-2 , Neoplasias Colorrectales/metabolismo , Células Epiteliales/metabolismo , Predisposición Genética a la Enfermedad , Humanos , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Neoplasias Peritoneales/metabolismo , Fenotipo , Estudios Prospectivos , Transducción de Señal , Factores de Tiempo , Factor de Crecimiento Transformador beta/metabolismo , Células Tumorales Cultivadas , Microambiente TumoralRESUMEN
Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis induce sustained clinical responses in a sizable minority of cancer patients. We found that primary resistance to ICIs can be attributed to abnormal gut microbiome composition. Antibiotics inhibited the clinical benefit of ICIs in patients with advanced cancer. Fecal microbiota transplantation (FMT) from cancer patients who responded to ICIs into germ-free or antibiotic-treated mice ameliorated the antitumor effects of PD-1 blockade, whereas FMT from nonresponding patients failed to do so. Metagenomics of patient stool samples at diagnosis revealed correlations between clinical responses to ICIs and the relative abundance of Akkermansia muciniphila Oral supplementation with A. muciniphila after FMT with nonresponder feces restored the efficacy of PD-1 blockade in an interleukin-12-dependent manner by increasing the recruitment of CCR9+CXCR3+CD4+ T lymphocytes into mouse tumor beds.
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Trasplante de Microbiota Fecal , Microbioma Gastrointestinal/inmunología , Inmunoterapia/métodos , Neoplasias/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Animales , Antibacterianos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD4/inmunología , Heces/microbiología , Microbioma Gastrointestinal/genética , Humanos , Interleucina-12/inmunología , Metagenoma/genética , Ratones , Receptores CCR/inmunología , Receptores CXCR3/inmunología , Linfocitos T/inmunología , Verrucomicrobia/genética , Verrucomicrobia/inmunologíaRESUMEN
Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder that typically associates with mutations in epigenetic, splicing, and signaling genes. Genetically modified mouse models only partially recapitulate the disease phenotype, whereas xenotransplantation of CMML cells in immunocompromised mice has been rarely successful so far. Here, CMML CD34+ cells sorted from patient bone marrow (BM) or peripheral blood (PB) were injected intravenously into NSG (NOD/LtSz-scid IL2rγnull) mice and NSG mice engineered to express human granulo-monocyte colony-stimulating factor, stem cell factor, and interleukin-3 (NSGS mice). Fifteen out of 16 patient samples (94%) successfully engrafted into NSG or NSGS or both mouse strains. The expansion of human cells, predominant in the BM, was also observed in the spleen and the PB and was greatly enhanced in mice producing the 3 human cytokines. Gene mutations identified in engrafted cells were mostly similar to those identified in patient cells before injection. Successful secondary engraftment was obtained in NSGS mice in 3 out of 10 attempts. Thus, primary CMML leukemic cells expand much better in NSGS compared with NSG mice with limited efficacy of secondary transplant.
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Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome that exhibits an erythroid-specific phenotype. In at least 70% of cases, DBA is related to a haploinsufficient germ line mutation in a ribosomal protein (RP) gene. Additional cases have been associated with mutations in GATA1. We have previously established that the RPL11+/Mut phenotype is more severe than RPS19+/Mut phenotype because of delayed erythroid differentiation and increased apoptosis of RPL11+/Mut erythroid progenitors. The HSP70 protein is known to protect GATA1, the major erythroid transcription factor, from caspase-3 mediated cleavage during normal erythroid differentiation. Here, we show that HSP70 protein expression is dramatically decreased in RPL11+/Mut erythroid cells while being preserved in RPS19+/Mut cells. The decreased expression of HSP70 in RPL11+/Mut cells is related to an enhanced proteasomal degradation of polyubiquitinylated HSP70. Restoration of HSP70 expression level in RPL11+/Mut cells reduces p53 activation and rescues the erythroid defect in DBA. These results suggest that HSP70 plays a key role in determining the severity of the erythroid phenotype in RP-mutation-dependent DBA.
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Hypomorphic mutation of apoptosis-inducing factor (AIF) in the whole body or organ-specific knockout of AIF compromises the activity of respiratory chain complexes I and IV, as it confers resistance to obesity and diabetes induced by high-fat diet. The mitochondrial defect induced by AIF deficiency can be explained by reduced AIF-dependent mitochondrial import of CHCHD4, which in turn is required for optimal import and assembly of respiratory chain complexes. Here we show that, as compared to wild type control littermates, mice with a heterozygous knockout of CHCHD4 exhibit reduced weight gain when fed with a Western style high-fat diet. This finding suggests widespread metabolic epistasis among AIF and CHCHD4. Targeting either of these proteins or their functional interaction might constitute a novel strategy to combat obesity.
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Factor Inductor de la Apoptosis/metabolismo , Dieta Alta en Grasa , Epistasis Genética/fisiología , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/deficiencia , Animales , Factor Inductor de la Apoptosis/genética , Dieta Alta en Grasa/métodos , Femenino , Ratones , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas del Complejo de Importación de Proteínas Precursoras MitocondrialesRESUMEN
Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, beyond its apoptotic function, is required for the normal expression of major respiratory chain complexes. Here we identified an AIF-interacting protein, CHCHD4, which is the central component of a redox-sensitive mitochondrial intermembrane space import machinery. Depletion or hypomorphic mutation of AIF caused a downregulation of CHCHD4 protein by diminishing its mitochondrial import. CHCHD4 depletion sufficed to induce a respiratory defect that mimicked that observed in AIF-deficient cells. CHCHD4 levels could be restored in AIF-deficient cells by enforcing its AIF-independent mitochondrial localization. This modified CHCHD4 protein reestablished respiratory function in AIF-deficient cells and enabled AIF-deficient embryoid bodies to undergo cavitation, a process of programmed cell death required for embryonic morphogenesis. These findings explain how AIF contributes to the biogenesis of respiratory chain complexes, and they establish an unexpected link between the vital function of AIF and the propensity of cells to undergo apoptosis.
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Factor Inductor de la Apoptosis/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Secuencia de Aminoácidos , Animales , Factor Inductor de la Apoptosis/genética , Línea Celular Tumoral , Transporte de Electrón/genética , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Humanos , Immunoblotting , Ratones Noqueados , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas/genética , Interferencia de ARN , Factores de TiempoRESUMEN
Hematopoietic stem cells (HSCs) are characterized by the capacity for self-renewal and the ability to reconstitute the entire hematopoietic compartment. Thrombopoietin maintains adult HSCs in a quiescent state through the induction of cell cycle inhibitors p57(Kip2) and p19(INK4d). Using the p19(INK4d-/-) mouse model, we investigated the role of p19(INK4d) in basal and stress-induced hematopoiesis. We demonstrate that p19(INK4d) is involved in the regulation of HSC quiescence by inhibition of the G0/G1 cell cycle transition. Under genotoxic stress conditions, the absence of p19(INK4d) in HSCs leads to accelerated cell cycle exit, accumulation of DNA double-strand breaks, and apoptosis when cells progress to the S/G2-M stages of the cell cycle. Moreover, p19(INK4d) controls the HSC microenvironment through negative regulation of megakaryopoiesis. Deletion of p19(INK4d) results in megakaryocyte hyperproliferation and increased transforming growth factor ß1 secretion. This leads to fibrosis in the bone marrow and spleen, followed by loss of HSCs during aging.
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Senescencia Celular/genética , Inhibidor p19 de las Quinasas Dependientes de la Ciclina/genética , Daño del ADN , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Nicho de Células Madre/genética , Animales , Médula Ósea/metabolismo , Médula Ósea/patología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Recuento de Células , Diferenciación Celular/genética , Linaje de la Célula/genética , Inhibidor p19 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p19 de las Quinasas Dependientes de la Ciclina/metabolismo , Hematopoyesis , Ratones , Ratones Noqueados , Osteosclerosis/genética , Osteosclerosis/patología , Mielofibrosis Primaria/genética , Mielofibrosis Primaria/patología , Fase de Descanso del Ciclo Celular/genética , Células del Estroma/metabolismoRESUMEN
In a previous study, we showed that cetuximab, a monoclonal antibody directed towards epidermal growth factor receptor, could inhibit P-glycoprotein (P-gp), an efflux protein of ATP-binding cassette family, and lead to an increased P-gp substrate intracellular concentration. Cetuximab is given with irinotecan to patients with metastasis colorectal cancer who did not respond to irinotecan-based therapy. The mechanism of this successful clinical reversion remains unknown. As irinotecan is a P-gp substrate, we tested here whether cetuximab could modify irinotecan concentration in mice. Therefore, concentrations of irinotecan and of its active metabolite SN-38 were measured by HPLC in plasma and tumour of mice bearing a human colorectal carcinoma xenograft when irinotecan is given orally alone or after a pretreatment with cetuximab. Pharmacokinetic analysis showed no significant modification of irinotecan concentrations but a significant increase (1.7-fold) in SN-38 AUCs in plasma and in tumour after a pretreatment with cetuximab. Those results suggest that cetuximab influence irinotecan distribution into tissues probably due to inhibition of P-gp. As SN-38 is 200-fold more potent than irinotecan, cetuximab could reverse irinotecan resistance by an effect on its active metabolite. Inhibiting SN-38 efflux by P-gp drug transporters in biliary system and tumour can lead to pharmacokinetic modification and a higher anticancer efficacy.
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Anticuerpos Monoclonales Humanizados/farmacología , Camptotecina/análogos & derivados , Neoplasias Colorrectales/tratamiento farmacológico , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Administración Oral , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Área Bajo la Curva , Transporte Biológico/efectos de los fármacos , Camptotecina/farmacocinética , Camptotecina/farmacología , Cetuximab , Neoplasias Colorrectales/patología , Interacciones Farmacológicas , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Irinotecán , Ratones , Ratones Desnudos , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
KRAS mutation is a negative predictive prognostic factor during metastatic colorectal cancer treatment with antiepidermal growth factor receptor antibodies. For affected patients, new therapeutics must be explored. Our objective was to study efficacy of two drugs with different mechanisms of action, everolimus (mTOR inhibitor) and lapatinib (tyrosine kinase inhibitor), in a mouse xenograft model. We chose a model obtained after engraftment of a tumor originating from a human tumor collection. The patient was affected by a metastasis colorectal carcinoma resistant to cetuximab with KRAS mutation. From a previous study in mice, we know that everolimus is a P-glycoprotein (P-gp) substrate and that a lapatinib pretreatment increases significantly (2.6-fold) everolimus AUC by inhibiting its intestinal P-gp efflux. We hence tested the effect of these drugs alone or combined. Mice bearing the xenografts were divided in four groups: control, lapatinib, everolimus, and L/E group (L/E: 2 days of lapatinib 200 mg/kg and then 3 days of everolimus 1 mg/kg). Tumor volumes and treatment toxicities were evaluated. Sixteen days after treatment initiation, the group L/E was the first one in which tumor volume average was significantly lower than the one of control group (193 ± 90 vs. 395 ± 171 mm(3) ; P = 0.0025). After 4 weeks of treatment, inhibition of tumor growth in lapatinib, everolimus, and L/E groups reached, respectively, 49, 53, and 57%. Each drug showed significant antitumor activity. Only moderate hematologic toxicity signs were observed. These results lead to new perspectives for new oral drugs in metastatic KRAS-mutated colorectal cancer resistant to standard chemotherapy.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Quinazolinas/farmacología , Sirolimus/análogos & derivados , Proteínas ras/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Resistencia a Antineoplásicos , Everolimus , Femenino , Humanos , Lapatinib , Ratones , Ratones Desnudos , Mutación , Quinazolinas/administración & dosificación , Sirolimus/administración & dosificación , Sirolimus/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/metabolismoRESUMEN
PURPOSE: Patient-derived xenograft models are considered to represent the heterogeneity of human cancers and advanced preclinical models. Our consortium joins efforts to extensively develop and characterize a new collection of patient-derived colorectal cancer (CRC) models. EXPERIMENTAL DESIGN: From the 85 unsupervised surgical colorectal samples collection, 54 tumors were successfully xenografted in immunodeficient mice and rats, representing 35 primary tumors, 5 peritoneal carcinoses and 14 metastases. Histologic and molecular characterization of patient tumors, first and late passages on mice includes the sequence of key genes involved in CRC (i.e., APC, KRAS, TP53), aCGH, and transcriptomic analysis. RESULTS: This comprehensive characterization shows that our collection recapitulates the clinical situation about the histopathology and molecular diversity of CRC. Moreover, patient tumors and corresponding models are clustering together allowing comparison studies between clinical and preclinical data. Hence, we conducted pharmacologic monotherapy studies with standard of care for CRC (5-fluorouracil, oxaliplatin, irinotecan, and cetuximab). Through this extensive in vivo analysis, we have shown the loss of human stroma cells after engraftment, observed a metastatic phenotype in some models, and finally compared the molecular profile with the drug sensitivity of each tumor model. Through an experimental cetuximab phase II trial, we confirmed the key role of KRAS mutation in cetuximab resistance. CONCLUSIONS: This new collection could bring benefit to evaluate novel targeted therapeutic strategies and to better understand the basis for sensitivity or resistance of tumors from individual patients.
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Transformación Celular Neoplásica/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales Humanizados , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Cetuximab , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Femenino , Fluorouracilo/administración & dosificación , Regulación Neoplásica de la Expresión Génica , Humanos , Irinotecán , Masculino , Ratones , Inestabilidad de Microsatélites , Persona de Mediana Edad , Estadificación de Neoplasias , Compuestos Organoplatinos/administración & dosificación , Oxaliplatino , RatasRESUMEN
Bone metastases have a devastating impact on quality of life and bone pain in patients with prostate cancer and decrease survival. Animal models are important tools in investigating the pathogenesis of the disease and in developing treatment strategies for bone metastases, but few animal models recapitulate spontaneous clinical bone metastatic spread. In the present study, IGR-CaP1, a new cell line derived from primary prostate cancer, was stably transduced with a luciferase-expressing viral vector to monitor tumor growth in mice using bioluminescence imaging. The IGR-CaP1 tumors grew when subcutaneously injected or when orthotopically implanted, reconstituted the prostate adenocarcinoma with glandular acini-like structures, and could disseminate to the liver and lung. Bone lesions were detected using bioluminescence imaging after direct intratibial or intracardiac injections. Anatomic bone structure assessed using high-resolution computed tomographic scans showed both lytic and osteoblastic lesions. Technetium Tc 99m methylene diphosphonate micro single-photon emission computed tomography confirmed the mixed nature of the lesions and the intensive bone remodeling. We also identified an expression signature for responsiveness of IGR-CaP1 cells to the bone microenvironment, namely expression of CXCR4, MMP-9, Runx2, osteopontin, osteoprotegerin, ADAMTS14, FGFBP2, and HBB. The IGR-CaP1 cell line is a unique model derived from a primary tumor, which can reconstitute human prostate adenocarcinoma in animals and generate experimental bone metastases, providing a novel means for understanding the mechanisms of bone metastasis progression and allowing preclinical testing of new therapies.
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Adenocarcinoma/patología , Neoplasias Óseas/secundario , Línea Celular Tumoral , Modelos Animales de Enfermedad , Neoplasias de la Próstata/patología , Adenocarcinoma/genética , Animales , Neoplasias Óseas/diagnóstico , Neoplasias Óseas/genética , Remodelación Ósea , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Osteólisis , Neoplasias de la Próstata/genética , Trasplante Heterólogo , Carga TumoralRESUMEN
Innovative cell-based therapies are considered as alternatives to liver transplantation. Recent progress in lentivirus-mediated hepatocyte transduction has renewed interest in cell therapy for the treatment of inherited liver diseases. However, hepatocyte transplantation is still hampered by inefficient hepatocyte engraftment. We previously showed that partial portal vein embolization (PVE) improved hepatocyte engraftment in a nonhuman primate model. We developed here an ex vivo approach based on PVE and lentiviral-mediated transduction of hepatocytes from normal (New Zealand White, NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits: the large animal model of familial hypercholesterolemia type IIa (FH). FH is a life-threatening human inherited autosomal disease caused by a mutation in the low-density lipoprotein receptor (LDLR) gene, which leads to severe hypercholesterolemia and premature coronary heart disease. Rabbit hepatocytes were isolated from the resected left liver lobe, and the portal branches of the median lobes were embolized with Histoacryl® glue under radiologic guidance. NZW and WHHL hepatocytes were each labeled with Hoechst dye or transduced with lentivirus expressing GFP under the control of a liver-specific promoter (mTTR, a modified murine transthyretin promoter) and were then immediately transplanted back into donor animals. In our conditions, 65-70% of the NZW and WHHL hepatocytes were transduced. Liver repopulation after transplantation with the Hoechst-labeled hepatocytes was 3.5 ± 2%. It was 1.4 ± 0.6% after transplantation with either the transduced NZW hepatocytes or the transduced WHHL hepatocytes, which was close to that obtained with Hoechst-labeled cells, given the mean transduction efficacy. Transgene expression persisted for at least 8 weeks posttransplantation. Transduction of WHHL hepatocytes with an LDLR-encoding vector resulted in phenotypic correction in vitro as assessed by internalization of fluorescent LDL ligands. In conclusion, our results have applications for the treatment of inherited metabolic liver diseases, such as FH, by transplantation of lentivirally transduced hepatocytes.
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RUNX1 encodes a DNA-binding α subunit of the core-binding factor, a heterodimeric transcription factor. RUNX1 is a master regulatory gene in hematopoiesis and its disruption is one of the most common aberrations in acute leukemia. Inactivating or dominant-negative mutations in the RUNX1 gene have been also identified in pedigrees of familial platelet disorders with a variable propensity to develop acute myeloid leukemia (FPD/AML). We performed analysis of hematopoiesis from 2 FPD/AML pedigrees with 2 distinct RUNX1 germline mutations, that is, the R139X in a pedigree without AML and the R174Q mutation in a pedigree with AML. Both mutations induced a marked increase in the clonogenic potential of immature CD34(+)CD38(-) progenitors, with some self-renewal capacities observed only for R174Q mutation. This increased proliferation correlated with reduction in the expression of NR4A3, a gene previously implicated in leukemia development. We demonstrated that NR4A3 was a direct target of RUNX1 and that restoration of NR4A3 expression partially reduced the clonogenic potential of patient progenitors. We propose that the down-regulation of NR4A3 in RUNX1-mutated hematopoietic progenitors leads to an increase in the pool of cells susceptible to be hit by secondary leukemic genetic events.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Hematopoyesis , Leucemia Mieloide Aguda/genética , Deficiencia de Almacenamiento del Pool Plaquetario/genética , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Adolescente , Adulto , Animales , Proliferación Celular , Células Cultivadas , Células Clonales/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Células HEK293 , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/fisiopatología , Masculino , Ratones , Persona de Mediana Edad , Mutación , Linaje , Deficiencia de Almacenamiento del Pool Plaquetario/metabolismo , Deficiencia de Almacenamiento del Pool Plaquetario/fisiopatología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/trasplante , Adulto JovenRESUMEN
Transforming growth factor-ß1 (TGF-ß1) is the most important cytokine involved in the promotion of myelofibrosis. Mechanisms leading to its local activation in the bone marrow environment remain unclear. As a recent study has highlighted the role of thrombospondin-1 (TSP-1) in platelet-derived TGF-ß1 activation, we investigated the role of TSP-1 in the TPO(high) murine model of myelofibrosis. Two groups of engrafted mice, WT TPO(high) and Tsp-1-null TPO(high), were constituted. All mice developed a similar myeloproliferative syndrome and an increase in total TGF-ß1 levels in the plasma and in extracellular fluids of marrow and spleen. Surprisingly, we were able to detect the active form of TGF-ß1 in Tsp-1-null TPO(high) mice. Accordingly, these mice developed marrow and spleen fibrosis, with intriguingly a higher grade than in WT TPO(high) mice. Our results show that TSP-1 is not the major activator of TGF-ß1 in TPO-induced myelofibrosis, suggesting the contribution of another mechanism in the megakaryocyte/platelet compartment.
Asunto(s)
Mielofibrosis Primaria/inducido químicamente , Mielofibrosis Primaria/patología , Trombopoyetina/efectos adversos , Trombospondina 1/fisiología , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Plaquetas/metabolismo , Plaquetas/patología , Médula Ósea/metabolismo , Médula Ósea/patología , Femenino , Masculino , Megacariocitos/metabolismo , Megacariocitos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mielofibrosis Primaria/metabolismo , Bazo/metabolismo , Bazo/patologíaRESUMEN
The aim of this study was to investigate the effect that recombinant interleukin-2 (rIL-2) (0.16 MUI/injection) had on the pharmacokinetics of imatinib (IM) in plasma. In this study, IM was given orally to mice at a dose of 150 mg/kg once a day for 11 days (from day 1 to 11) either alone or in combination with intraperitoneal injections of rIL-2 twice a day from day 8 to 11. Pharmacokinetic parameters were determined using WinNonLin software. Areas under the curve were compared using Bailer's method. The repeated administration of the rIL-2+IM combination was shown to have two pharmacokinetic advantages compared with repeated IM doses alone. In addition to the pharmacodynamic interest of this treatment, we found that the combined treatment significantly increased the IM Cmax (P<0.05) and significantly increased the IM trough concentration (C(24 h)) (P<0.01), which was always above the minimum therapeutic IM concentration (1 mumol/l) in plasma. Those pharmacokinetic modifications may be explained, in part, by a decrease in the P-glycoprotein expression in the three intestinal segments of the mice (duodenum, P<0.01; jejunum, P<0.05; and ileum, P<0.05) and a decrease in BCRP expression in the duodenum segment (P<0.05) due to rIL-2. In another experiment, we found a significant induction of intestinal P-glycoprotein expression in mice that had been given IM orally (150 mg/kg) twice a day for 11 days. It would be interesting to further investigate the IM disposition associated with rIL-2 treatment for clinical applications.