RESUMO
BACKGROUND: Recent technical progress makes sophisticated noninvasive imaging methods available for murine models. For the first time, in this study, we applied fluorodeoxyglucose (FDG)-positron emission tomography (PET)-CT and FDG-PET-MRI to a murine orthotopic model of head and neck cancer immunotherapy. METHODS: Tumor growth of floor of the mouth tumors was evaluated by multimodal small-animal imaging using FDG-PET-CT and FDG-PET-MRI. The immunotherapeutic effects of anti-CD137 antibody therapy were examined on body weight, tumor growth, and tumor-infiltrating immune cells in longitudinal imaging studies and immunohistochemical analyses. RESULTS: Imaging revealed aggressive, fast-growing tumors without evidence of local or distant metastases. CD137 immunotherapy decreased tumor take and growth and stabilized body weight over time. A clear case of tumor regression was demonstrated by longitudinal PET-CT. CONCLUSION: The murine model mimics the characteristics of head and neck cancer in humans and offers excellent opportunities to investigate immunomodulatory anticancer drugs. The CD137 antibody showed antitumor effects in some therapy-responsive mice.
Assuntos
Ligante 4-1BB/antagonistas & inibidores , Carcinoma de Células Escamosas/diagnóstico por imagem , Modelos Animais de Doenças , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Imunoterapia/métodos , Neoplasias Bucais/diagnóstico por imagem , Neoplasias Bucais/terapia , Imagem Multimodal/métodos , Animais , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/terapia , Linhagem Celular Tumoral , Feminino , Fluordesoxiglucose F18 , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/terapia , Imuno-Histoquímica , Estudos Longitudinais , Imageamento por Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos C3H , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
For preclinical studies of immune-modulating anticancer drugs a murine model that attempts to parallel the clinical nature of head and neck cancer in fully immunocompetent mice is required. In this study we compared features of the squamous cell carcinoma (SCC) VII model after subcutaneous (back, flank) and orthotopic (floor of mouth) injection both in fully immunocompetent C3H/HeN and in previously studied C3H/HeJ mice, which harbor a functional toll-like receptor 4 (TLR-4) deficiency. As C3H/HeN mice do not harbor this deficiency, the presented murine model is an optimization of previously described C3H/HeJ models, which, because of the TLR-4-deficiency, have inherent drawbacks for tumor immunologic studies. We found that tumor growth was accelerated and tumor incidence was increased by about 20% after s.c. injection in TLR-4-deficient mice. Strikingly, tumor-related weight loss (cachexia) was more pronounced in fully immunocompetent C3H/HeN mice (26%) versus TLR-4-deficient C3H/HeJ mice (7.9% weight loss) at high tumor dose. Orthotopic tumors were biologically distinct from subcutaneous tumors as they showed accelerated growth and a distinct immune cell infiltrate. We conclude that a model of orthotopic implantation of SCC VII tumor cells into fully immunocompetent syngeneic C3H/HeN mice reflects features of human head and neck cancer and provides a valuable experimental model for immunological studies in this tumor entity. Our data suggest that TLR-4 expressed by host cells is involved in the regulation of tumor-related cachexia and tumor control.
Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/patologia , Imunocompetência , Receptor 4 Toll-Like/fisiologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Caquexia/genética , Caquexia/metabolismo , Caquexia/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Transplante de Neoplasias , Receptores de Quimiocinas/metabolismo , Fatores de Tempo , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Carga TumoralRESUMO
RATIONALE: Congestive heart failure (CHF) frequently results in remodeling and increased tone of pulmonary resistance vessels. This adaptive response, which aggravates pulmonary hypertension and thus, promotes right ventricular failure, has been attributed to lung endothelial dysfunction. OBJECTIVE: We applied real-time fluorescence imaging to identify endothelial dysfunction and underlying molecular mechanisms in an experimental model of CHF induced by supracoronary aortic banding in rats. METHODS AND RESULTS: Endothelial dysfunction was evident in lungs of CHF rats as impaired endothelium-dependent vasodilation and lack of endothelial NO synthesis in response to mechanical stress, acetylcholine, or histamine. This effect was not attributable to downregulation of endothelial NO synthase. Imaging of the cytosolic Ca(2+) concentration ([Ca(2+)](i)) revealed a singular impairment of endothelial [Ca(2+)](i) homeostasis and signaling characterized by a lack of [Ca(2+)](i) oscillations and deficient or attenuated [Ca(2+)](i) responses to mechanical stress, histamine, acetylcholine, or thapsigargin. Reconstitution of a [Ca(2+)](i) signal by ionophore treatment restored endothelial NO production, but lack of endothelial responsiveness was not primarily attributable to downregulation of Ca(2+) influx channels in CHF. Rather, we identified a massive remodeling of the endothelial cytoskeleton in the form of an increased expression of beta-actin and F-actin formation which contributed critically to endothelial dysfunction in CHF because cytoskeletal disruption by cytochalasin D largely reconstituted endothelial [Ca(2+)](i) signaling and NO production. CONCLUSIONS: Our findings characterize a unique scenario of endothelial dysfunction in CHF that is caused by a singular impairment of [Ca(2+)](i) signaling, and identify cytoskeletal reorganization as a major regulator of endothelial signaling and function.
Assuntos
Sinalização do Cálcio , Citoesqueleto/metabolismo , Endotélio Vascular/metabolismo , Insuficiência Cardíaca/complicações , Hipertensão Pulmonar/etiologia , Pulmão/irrigação sanguínea , Vasodilatação , Acetilcolina/farmacologia , Actinas/metabolismo , Animais , Pressão Sanguínea , Sinalização do Cálcio/efeitos dos fármacos , Antagonistas Colinérgicos/farmacologia , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Histamina/farmacologia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Ionóforos/farmacologia , Masculino , Microscopia de Fluorescência , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/metabolismo , Perfusão , Ratos , Ratos Sprague-Dawley , Estresse Mecânico , Fatores de Tempo , Canais de Potencial de Receptor Transitório/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
OBJECTIVES: MR techniques have been reported as an alternative to conventional sialography. High-field systems (7 T) provide new contrasts coupled with increased signal-to-noise ratio, and hence higher spatial resolution. To our knowledge, no measurements of the parotid gland at 7 T have been reported. Therefore, our study aimed to optimize sequences for high-field MR imaging of the parotid gland and duct, as well as the facial nerve at 7 T and show the potential of high field imaging. MATERIALS AND METHODS: A 7 T whole-body scanner was used together with a 10-cm-diameter loop coil. Various GRE (MEDIC, DESS) and TSE (PD/T2, STIR) sequences were optimized and subsequently tested on 4 healthy volunteers and 4 patients. High-resolution images were compared with 1.5 T images both quantitatively (signal-to-noise ratio, contrast-to-noise) and qualitatively (visual rating of 2 independent readers). RESULTS: The high 0.6 mm isotropic resolution of the 3D DESS sequence was very useful for defining an oblique orientation with most of the duct being in-plane for subsequent imaging. With the MEDIC sequence, very fine branches of the duct were visible; furthermore, MEDIC yielded a very good depiction of lymph nodes. Severe specific absorption rate problems were observed with the STIR sequence at 7 T. Gland tissue in tumor patients can be well characterized with the PD/T2 TSE. Highest contrast-to-noise between duct and gland was achieved with the 7 T DESS. At 1.5 T, only the STIR sequence showed comparable quality to the overall superiority of the 7 T sequences. The facial nerve could only be depicted close to the skull base. CONCLUSION: MR imaging at 7 T provides excellent image contrast and resolution of the parotid gland and duct. The proposed protocol offers a noninvasive examination within about 30 minutes.
Assuntos
Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Imageamento por Ressonância Magnética/métodos , Glândula Parótida/anatomia & histologia , Reconhecimento Automatizado de Padrão/métodos , Adulto , Humanos , Masculino , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Lung capillary endothelial cells (ECs) are a critical target of oxygen toxicity and play a central role in the pathogenesis of hyperoxic lung injury. To determine mechanisms and time course of EC activation in normobaric hyperoxia, we measured endothelial concentration of reactive oxygen species (ROS) and cytosolic calcium ([Ca(2+)](i)) by in situ imaging of 2',7'-dichlorofluorescein (DCF) and fura 2 fluorescence, respectively, and translocation of the small GTPase Rac1 by immunofluorescence in isolated perfused rat lungs. Endothelial DCF fluorescence and [Ca(2+)](i) increased continuously yet reversibly during a 90-min interval of hyperoxic ventilation with 70% O(2), demonstrating progressive ROS generation and second messenger signaling. ROS formation increased exponentially with higher O(2) concentrations. ROS and [Ca(2+)](i) responses were blocked by the mitochondrial complex I inhibitor rotenone, whereas inhibitors of NAD(P)H oxidase and the intracellular Ca(2+) chelator BAPTA predominantly attenuated the late phase of the hyperoxia-induced DCF fluorescence increase after > 30 min. Rac1 translocation in lung capillary ECs was barely detectable at normoxia but was prominent after 60 min of hyperoxia and could be blocked by rotenone and BAPTA. We conclude that hyperoxia induces ROS formation in lung capillary ECs, which initially originates from the mitochondrial electron transport chain but subsequently involves activation of NAD(P)H oxidase by endothelial [Ca(2+)](i) signaling and Rac1 activation. Our findings demonstrate rapid activation of ECs by hyperoxia in situ and identify mechanisms that may be relevant in the initiation of hyperoxic lung injury.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Hiperóxia/metabolismo , Pulmão/irrigação sanguínea , Oxigênio/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Cálcio/fisiologia , Capilares/metabolismo , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Ativação Enzimática , Fluoresceínas , Corantes Fluorescentes , Fura-2 , Hiperóxia/patologia , Técnicas In Vitro , Masculino , Microcirculação/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/fisiologia , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Whereas endothelial responses to shear stress have been studied extensively, the responses to circumferential vascular stretch are yet poorly defined. Circumferential stretch in pulmonary microvessels is largely determined by the transmural pressure gradient, hence by both vascular perfusion and alveolar ventilation pressures. Here, we have studied the production of nitric oxide (NO) by the endothelial nitric oxide synthase (eNOS) in two different models of vascular stretch in the intact lung: In isolated-perfused rat lungs, vascular stretch was induced by elevation of vascular pressure. In situ digital fluorescence microscopy revealed stretch-dependent NO production, which was localized to capillary endothelial cells and inhibited by NOS blockers. In isolated-perfused mouse lungs, vascular stretch was generated by ventilation with elevated negative pressure. Stretch-induced phosphorylation of Akt and eNOS in lung endothelial cells was demonstrated by immunohistochemistry and increased NO production by in situ fluorescence microscopy. Stretch-induced endothelial responses in both models were abrogated by pretreatment with phosphatidylinositol-3-OH kinase inhibitors. These findings demonstrate that circumferential stretch activates NO production in pulmonary endothelial cells by a signaling cascade involving phosphatidylinositol-3-OH kinase, Akt, and eNOS and that this response is independent from the mechanical factors causing vascular distension.