RESUMEN
Genetically engineered mouse models (GEMMs) of lung cancer closely recapitulate the human disease but suffer from the difficulty of evaluating tumor growth by conventional methods. Herein, a novel automated image analysis method for estimating the lung tumor burden from in vivo micro-computed tomography (micro-CT) data is described. The proposed tumor burden metric is the segmented soft tissue volume contained within a chest space region of interest, excluding an estimate of the heart volume. The method was validated by comparison with previously published manual analysis methods and applied in two therapeutic studies in a mutant K-ras GEMM of non-small cell lung carcinoma. Mice were imaged by micro-CT pre-treatment and stratified into four treatment groups: an antibody inhibiting vascular endothelial growth factor (anti-VEGF), chemotherapy, combination of anti-VEGF and chemotherapy, or control antibody. In the first study, post-treatment imaging was performed 4 weeks later. In the second study, mice were scanned serially on a high-throughput scanner every 2 weeks for 8 weeks during treatment. In both studies, the automated tumor burden estimates were well correlated with manual metrics (r value range: 0.83-0.93, P < .0001) and showed a similar, significant reduction in tumor growth in mice treated with anti-VEGF alone or in combination with chemotherapy. Given the fully automated nature of this technique, the proposed analysis method can provide a valuable tool in preclinical drug research for screening and randomizing animals into treatment groups and evaluating treatment efficacy in mouse models of lung cancer in a highly robust and efficient manner.
RESUMEN
Activating mutations in the KRAS oncogene occur in approximately 90% of pancreatic cancers, resulting in aberrant activation of the MAPK and the PI3K pathways, driving malignant progression. Significant efforts to develop targeted inhibitors of nodes within these pathways are underway and several are currently in clinical trials for patients with KRAS-mutant tumors, including patients with pancreatic cancer. To model MEK and PI3K inhibition in late-stage pancreatic cancer, we conducted preclinical trials with a mutant Kras-driven genetically engineered mouse model that faithfully recapitulates human pancreatic ductal adenocarcinoma development. Treatment of advanced disease with either a MEK (GDC-0973) or PI3K inhibitor (GDC-0941) alone showed modest tumor growth inhibition and did not significantly enhance overall survival. However, combination of the two agents resulted in a significant survival advantage as compared with control tumor-bearing mice. To model the clinical scenario, we also evaluated the combination of these targeted agents with gemcitabine, the current standard-of-care chemotherapy for pancreatic cancer. The addition of MEK or PI3K inhibition to gemcitabine, or the triple combination regimen, incrementally enhanced overall survival as compared with gemcitabine alone. These results are reminiscent of the survival advantage conferred in this model and in patients by the combination of gemcitabine and erlotinib, an approved therapeutic regimen for advanced nonresectable pancreatic cancer. Taken together, these data indicate that inhibition of MEK and PI3K alone or in combination with chemotherapy do not confer a dramatic improvement as compared with currently available therapies for patients with pancreatic cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Carcinoma Ductal Pancreático/tratamiento farmacológico , Desoxicitidina/análogos & derivados , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/administración & dosificación , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Azetidinas/administración & dosificación , Azetidinas/farmacología , Carcinoma Ductal Pancreático/genética , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Indazoles/administración & dosificación , Indazoles/farmacología , Ratones , Modelos Biológicos , Mutación , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Piperidinas/administración & dosificación , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/genética , Nivel de Atención , Sulfonamidas/administración & dosificación , Sulfonamidas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Priming of the organ-specific premetastatic sites is thought to be an important yet incompletely understood step during metastasis. In this study, we show that the metastatic tumors we examined overexpress granulocyte-colony stimulating factor (G-CSF), which expands and mobilizes Ly6G+Ly6C+ granulocytes and facilitates their subsequent homing at distant organs even before the arrival of tumor cells. Moreover, G-CSF-mobilized Ly6G+Ly6C+ cells produce the Bv8 protein, which has been implicated in angiogenesis and mobilization of myeloid cells. Anti-G-CSF or anti-Bv8 antibodies significantly reduced lung metastasis. Transplantation of Bv8 null fetal liver cells into lethally irradiated hosts also reduced metastasis. We identified an unexpected role for Bv8: the ability to stimulate tumor cell migration through activation of one of the Bv8 receptors, prokineticin receptor (PKR)-1. Finally, we show that administration of recombinant G-CSF is sufficient to increase the numbers of Ly6G+Ly6C+ cells in organ-specific metastatic sites and results in enhanced metastatic ability of several tumors.
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Antígenos Ly/inmunología , Factor Estimulante de Colonias de Granulocitos/farmacología , Granulocitos/efectos de los fármacos , Granulocitos/inmunología , Neoplasias Pulmonares/patología , Metástasis de la Neoplasia , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Movimiento Celular , Femenino , Perfilación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos/genética , Granulocitos/citología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones SCID , Análisis por Micromatrices , Trasplante de Neoplasias , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaRESUMEN
The low rate of approval of novel anti-cancer agents underscores the need for better preclinical models of therapeutic response as neither xenografts nor early-generation genetically engineered mouse models (GEMMs) reliably predict human clinical outcomes. Whereas recent, sporadic GEMMs emulate many aspects of their human disease counterpart more closely, their ability to predict clinical therapeutic responses has never been tested systematically. We evaluated the utility of two state-of-the-art, mutant Kras-driven GEMMs--one of non-small-cell lung carcinoma and another of pancreatic adenocarcinoma--by assessing responses to existing standard-of-care chemotherapeutics, and subsequently in combination with EGFR and VEGF inhibitors. Standard clinical endpoints were modeled to evaluate efficacy, including overall survival and progression-free survival using noninvasive imaging modalities. Comparisons with corresponding clinical trials indicate that these GEMMs model human responses well, and lay the foundation for the use of validated GEMMs in predicting outcome and interrogating mechanisms of therapeutic response and resistance.
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Modelos Animales de Enfermedad , Ingeniería Genética , Mutación/genética , Neoplasias/genética , Neoplasias/terapia , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Clorhidrato de Erlotinib , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Quinazolinas/uso terapéutico , Análisis de Supervivencia , Factor A de Crecimiento Endotelial Vascular/inmunología , GemcitabinaRESUMEN
In response to inflammatory stimuli, dendritic cells (DCs) trigger the process of maturation, a terminal differentiation program required to initiate T-lymphocyte responses. A hallmark of maturation is down-regulation of endocytosis, which is widely assumed to restrict the ability of mature DCs to capture and present antigens encountered after the initial stimulus. We found that mature DCs continue to accumulate antigens, especially by receptor-mediated endocytosis and phagocytosis. Internalized antigens are transported normally to late endosomes and lysosomes, loaded onto MHC class II molecules (MHCII), and then presented efficiently to T cells. This occurs despite the fact that maturation results in the general depletion of MHCII from late endocytic compartments, with MHCII enrichment being typically thought to be a required feature of antigen processing and peptide loading compartments. Internalized antigens can also be cross-presented on MHC class I molecules, without any reduction in efficiency relative to immature DCs. Thus, although mature DCs markedly down-regulate their capacity for macropinocytosis, they continue to capture, process, and present antigens internalized via endocytic receptors, suggesting that they may continuously initiate responses to newly encountered antigens during the course of an infection.
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Células Dendríticas/inmunología , Endocitosis , Receptores Inmunológicos/inmunología , Animales , Citometría de Flujo , Antígenos de Histocompatibilidad Clase II/inmunología , Ratones , Microscopía Electrónica , Microscopía Fluorescente , FagocitosisRESUMEN
The hepatocyte growth factor (HGF) and its receptor, c-Met, have been implicated in driving proliferation, invasion, and poor prognosis in pancreatic cancer. Here, we investigated the expression of HGF and c-Met in primary pancreatic cancers and described in vitro and in vivo models in which MetMAb, a monovalent antibody against c-Met, was evaluated. First, expression of HGF and MET mRNA was analyzed in 59 primary pancreatic cancers and 51 normal samples, showing that both factors are highly expressed in pancreatic cancer. We next examined HGF responsiveness in pancreatic cancer lines to select lines that proliferate in response to HGF. Based on these studies, two lines were selected for further in vivo model development: BxPC-3 (c-Met(+), HGF(-)) and KP4 (c-Met(+), HGF(+)) cells. As BxPC-3 cells are responsive to exogenous HGF, s.c. tumor xenografts were grown in a paracrine manner with purified human HGF provided by osmotic pumps, wherein MetMAb treatment significantly inhibited tumor growth. KP4 cells are autocrine for HGF and c-Met, and MetMAb strongly inhibited s.c. tumor growth. To better model pancreatic cancer and to enable long-term survival studies, an orthotopic model of KP4 was established. MetMAb significantly inhibited orthotopic KP4 tumor growth in 4-week studies monitored by ultrasound and also improved survival in 90-day studies. MetMAb significantly reduced c-Met phosphorylation in orthotopic KP4 tumors with a concomitant decrease in Ki-67 staining. These data suggest that the HGF/c-Met axis plays an important role in the progression of pancreatic cancer and that targeting c-Met therein may have therapeutic value.
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Adenocarcinoma/patología , Anticuerpos Monoclonales/inmunología , Neoplasias Pancreáticas/patología , Proteínas Tirosina Quinasas Receptoras/inmunología , Adenocarcinoma/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Inmunohistoquímica , Inmunoprecipitación , Ratones , Ratones Desnudos , Neovascularización Patológica/inmunología , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/metabolismo , Fosforilación , Transducción de Señal , Análisis de SupervivenciaRESUMEN
Magnetic resonance microscopy (microMRI) is becoming an important tool for non-destructive analysis of fixed brain tissue. However, unlike MRI, X-ray computed tomography (CT) scans show little native soft tissue contrast. In this paper, we explored the use of contrast enhanced (brains immersion stained in iodinated CT contrast media) micro-CT (microCT) for high resolution 3D imaging of fixed normal and pathological brains, compared to microMRI and standard histopathology. An optimum iodine concentration of 0.27 M resulted in excellent contrast between gray and white matter in normal brain and a wide range of anatomical structures were identified. In glioma bearing mouse brains, there was clear deliniation of tumor margin which closely matched that seen on histopathology sections. microCT tumor volume was strongly correlated with histopathology volume. Our data suggests that microCT image contrast in the immersion-stained brains is related to axonal density and myelin content. Compared to traditional histopathology, our microCT approach is relatively rapid and less labor intensive. In addition, compared to microMRI, microCT is robust and requires much lower equipment and maintenance costs. For simple measurements, such as tumor volume and non-destructive postmortem brain screening, microCT may prove to be a valuable alternative to standard histopathology or microMRI.
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Neoplasias Encefálicas/patología , Glioma/patología , Imagenología Tridimensional , Cambios Post Mortem , Tomografía Computarizada por Rayos X/métodos , Animales , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones Desnudos , ConejosRESUMEN
BACKGROUND: Heparan sulfate proteoglycans (HSPGs) use highly sulfated polysaccharide side-chains to interact with several key growth factors and morphogens, thereby regulating their accessibility and biological activity. Various sulfotransferases and sulfatases with differing specificities control the pattern of HSPG sulfation, which is functionally critical. Among these enzymes in the mouse are two secreted 6-O-endosulfatases, Sulf1 and Sulf2, which modify HSPGs in the extracellular matrix and on the cell surface. The roles of Sulf1 and Sulf2 during normal development are not well understood. METHODS/RESULTS: To investigate the importance of Sulf1 and Sulf2 for embryonic development, we generated mice genetically deficient in these genes and assessed the phenotypes of the resulting secreted sulfatase-deficient mice. Surprisingly, despite the established crucial role of HSPG interactions during development, neither Sulf1- nor Sulf2-deficient mice showed significant developmental flaws. In contrast, mice deficient in both Sulf1and Sulf2 exhibited highly penetrant neonatal lethality. Loss of viability was associated with multiple, although subtle, developmental defects, including skeletal and renal abnormalities. CONCLUSIONS: These results show that Sulf1 and Sulf2 play overlapping yet critical roles in mouse development and are redundant and essential for neonatal survival.