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Endothelial Notch signaling is critical for tumor angiogenesis. Notch1 blockade can interfere with tumor vessel function but causes tissue hypoxia and gastrointestinal toxicity. Notch4 is primarily expressed in endothelial cells, where it may promote angiogenesis; however, effective therapeutic targeting of Notch4 has not been successful. We developed highly specific Notch4-blocking antibodies, 6-3-A6 and humanized E7011, allowing therapeutic targeting of Notch4 to be assessed in tumor models. Notch4 was expressed in tumor endothelial cells in multiple cancer models, and endothelial expression was associated with response to E7011/6-3-A6. Anti-Notch4 treatment significantly delayed tumor growth in mouse models of breast, skin, and lung cancers. Enhanced tumor inhibition occurred when anti-Notch4 treatment was used in combination with chemotherapeutics. Endothelial transcriptomic analysis of murine breast tumors treated with 6-3-A6 identified significant changes in pathways of vascular function but caused only modest change in canonical Notch signaling. Analysis of early and late treatment timepoints revealed significant differences in vessel area and perfusion in response to anti-Notch4 treatment. We conclude that targeting Notch4 improves tumor growth control through endothelial intrinsic mechanisms. SIGNIFICANCE: A first-in-class anti-Notch4 agent, E7011, demonstrates strong antitumor effects in murine tumor models including breast carcinoma. Endothelial Notch4 blockade reduces perfusion and vessel area.
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Anticuerpos Neutralizantes , Neovascularización Patológica , Receptor Notch4 , Animales , Receptor Notch4/metabolismo , Ratones , Humanos , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Neovascularización Patológica/metabolismo , Femenino , Anticuerpos Neutralizantes/farmacología , Anticuerpos Neutralizantes/uso terapéutico , Línea Celular Tumoral , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Proliferación Celular/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismoRESUMEN
Pulmonary anthrax caused by exposure to inhaled Bacillus anthracis, the most lethal form of anthrax disease, is a continued military and public health concern for the United States. The vaccine AV7909, consisting of the licensed anthrax drug substance AVA adjuvanted with CpG7909, induces high levels of toxin neutralizing antibodies in healthy adults using fewer doses than AVA. This study compares the ability of one- or two-dose regimens of AV7909 to induce a protective immune response in guinea pigs challenged with a lethal dose of aerosolized B. anthracis spores 6 weeks after the last vaccine dose. The results indicated that AV7909 was less effective when delivered as a single dose compared to the two-dose regimen that resulted in dose-dependent protection against death. The toxin neutralizing assay (TNA) titer and anti-PA IgG responses were proportional to the protective efficacy, with a 50% TNA neutralizing factor (NF50) greater than 0.1 associated with survival in animals receiving two doses of vaccine. The strong protection at relatively low TNA NF50 titers in this guinea pig model supports the exploration of lower doses in clinical trials to determine if these protective levels of neutralizing antibodies can be achieved in humans; however, protection with a single dose may not be feasible.
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Vacunas contra el Carbunco , Carbunco , Bacillus anthracis , Adulto , Humanos , Animales , Cobayas , Carbunco/prevención & control , Anticuerpos Antibacterianos , Anticuerpos Neutralizantes , Antígenos BacterianosRESUMEN
Type I interferon-mediated activation of immune cells can facilitate the generation of productive tumor antigen-specific T cell responses in solid tumors. The cGAS/STING DNA sensing pathway is a critical upstream mediator of type I interferon production and is an important regulator of anti-tumor immunity. Numerous STING pathway agonists are now being tested in clinical trials, but the effectiveness of this approach is not yet clear and a better understanding of the relative importance of this pathway in various tumor settings is needed. We have evaluated syngeneic tumor models with different baseline inflammatory states to determine the contributions of STING activity in both tumor and non-tumor cellular compartments to anti-tumor immune responses. We find that productive anti-tumor immune responses in the poorly immunogenic B16F10 model show a strong dependence on STING expression in non-tumor cells. In the immunogenic MC38 model, constitutive STING activation in tumor cells can partially bypass the requirement for STING-dependent activity from immune cells. Our findings reveal multiple, context-dependent roles for STING activity in the regulation of anti-tumor immunity and the response to immunotherapy. In preclinical models where STING is basally active, checkpoint inhibition is more likely to have a therapeutic effect and removal of STING signaling from either the tumor or the non-tumor compartment has a minimal effect. Removal of STING signaling in both, however, diminishes the efficacy derived from checkpoint therapy. Further work is needed to understand the heterogeneity of STING signaling in patients, both in tumor cells and the tumor microenvironment, and the best means of harnessing this pathway to generate anti-tumor immunity and improve therapeutic outcomes.
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Interferón Tipo I , Neoplasias , Humanos , ADN , Inmunidad Innata , Inmunoterapia , Transducción de Señal , Microambiente TumoralRESUMEN
BACKGROUND: Bispecific T-cell engager (BiTE) molecules induce redirected lysis of cancer cells by T cells and are an emerging modality for solid tumor immunotherapy. While signs of clinical activity have been demonstrated, efficacy of T-cell engagers (TCEs) in solid tumors settings, molecular determinants of response, and underlying mechanisms of resistance to BiTE therapy require more investigation. METHODS: To uncover cancer cell-intrinsic genetic modifiers of TCE-mediated cytotoxicity, we performed genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loss-of-function and CRISPRa (CRISPR activation) gain-of-function screens using TCEs against two distinct tumor-associated antigens (TAAs). By using in vitro T-cell cytotoxicity assays and in vivo efficacy studies, we validated the roles of two common pathways identified in our screen, T-cell costimulation pathway and apoptosis pathway, as key modifiers of BiTE activity. RESULTS: Our genetic screens uncovered TAAs-independent cancer cell-intrinsic genes with functions in autophagy, T-cell costimulation, the apoptosis pathway, chromatin remodeling, and cytokine signaling that altered responsiveness to BiTE-mediated killing. Notably, loss of CD58 (the ligand of the CD2 T-cell costimulatory receptor), a gene frequently altered in cancer, led to decreased TCE-mediated cytotoxicity, T-cell activation and antitumor efficacy in vitro and in vivo. Moreover, the effects of CD58 loss were synergistically compounded by concurrent loss of CD80/CD86 (ligands for the CD28 T-cell costimulatory receptor), whereas joint CD2 and CD28 costimulation additively enhanced TCE-mediated killing, indicating non-redundant costimulatory mechanisms between the two pathways. Additionally, loss of CFLAR (Caspase-8 and FADD Like Apoptosis Regulator), BCL2L1, and BID (BH3 Interacting Domain Death Agonist) induced profound changes in sensitivity to TCEs, indicating that key regulators of apoptosis, which are frequently altered in cancer, impact tumor responsiveness to BiTE therapy. CONCLUSIONS: This study demonstrates that genetic alterations central to carcinogenesis and commonly detected in cancer samples lead to significant modulation of BiTE antitumor activity in vitro and in vivo, findings with relevance for a better understanding of patient responses to BiTE therapy and novel combinations that enhance TCE efficacy.
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Antígenos CD58/inmunología , Inmunoterapia , Neoplasias , Apoptosis , Antígenos CD28/metabolismo , Humanos , Activación de Linfocitos , Neoplasias/metabolismo , Linfocitos TRESUMEN
Therapeutic approaches are needed to promote T cell-mediated destruction of poorly immunogenic, "cold" tumors typically associated with minimal response to immune checkpoint blockade (ICB) therapy. Bispecific T cell engager (BiTE) molecules induce redirected lysis of cancer cells by polyclonal T cells and have demonstrated promising clinical activity against solid tumors in some patients. However, little is understood about the key factors that govern clinical responses to these therapies. Using an immunocompetent mouse model expressing a humanized CD3ε chain (huCD3e mice) and BiTE molecules directed against mouse CD19, mouse CLDN18.2, or human EPCAM antigens, we investigated the pharmacokinetic and pharmacodynamic parameters and immune correlates associated with BiTE efficacy across multiple syngeneic solid-tumor models. These studies demonstrated that pretreatment tumor-associated T cell density is a critical determinant of response to BiTE therapy, identified CD8+ T cells as important targets and mediators of BiTE activity, and revealed an antagonistic role for CD4+ T cells in BiTE efficacy. We also identified therapeutic combinations, including ICB and 4-1BB agonism, that synergized with BiTE treatment in poorly T cell-infiltrated, immunotherapy-refractory tumors. In these models, BiTE efficacy was dependent on local expansion of tumor-associated CD8+ T cells, rather than their recruitment from circulation. Our findings highlight the relative contributions of baseline T cell infiltration, local T cell proliferation, and peripheral T cell trafficking for BiTE molecule-mediated efficacy, identify combination strategies capable of overcoming resistance to BiTE therapy, and have clinical relevance for the development of BiTE and other T cell engager therapies.
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Anticuerpos Biespecíficos , Neoplasias , Animales , Anticuerpos Biespecíficos/uso terapéutico , Antígenos CD19 , Complejo CD3 , Linfocitos T CD8-positivos , Claudinas , Humanos , Inmunoterapia , Ratones , Neoplasias/tratamiento farmacológicoRESUMEN
Aortic stiffening, assessed as pulse-wave velocity (PWV), increases with age and is an important antecedent to, and independent predictor of, cardiovascular diseases (CVD) and other clinical disorders of aging. Aerobic exercise promotes lower levels of aortic stiffness in older adults, but the underlying mechanisms are incompletely understood, largely due to inherent challenges of mechanistic studies of large elastic arteries in humans. Voluntary wheel running (VWR) is distinct among experimental animal exercise paradigms in that it allows investigation of the physiologic effects of aerobic training without potential confounding influences of aversive molecular signaling related to forced exercise. In this study, we investigated whether VWR in mice may be a suitable model for mechanistic studies (i.e., "reverse translation") of the beneficial effects of exercise on arterial stiffness in humans. We found that 10 weeks of VWR in old mice (~ 28 months) reversed age-related elevations in aortic PWV assessed in vivo (Old VWR: 369 ± 19 vs. old sedentary: 439 ± 20 cm/s, P < 0.05). The de-stiffening effects of VWR were accompanied by normalization of age-related increases in ex vivo mechanical stiffness of aortic segments and aortic accumulation of collagen-I and advanced glycation end products, as well as lower levels of aortic superoxide and nitrotyrosine. Our results suggest that late-life VWR in mice recapitulates the aortic de-stiffening effects of exercise in humans and indicates important mechanistic roles for decreased oxidative stress and extracellular matrix remodeling. Therefore, VWR is a suitable model for further study of the mechanisms underlying beneficial effects of exercise on arterial stiffness.
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Rigidez Vascular , Animales , Aorta , Arterias , Ratones , Actividad Motora , Análisis de la Onda del PulsoRESUMEN
Bone marrow chimeras represent a key tool employed to understand biological contributions stemming from the hematopoietic versus the stromal compartment. In most institutions, cesium irradiators are used to lethally irradiate recipient animals prior to the injection of donor bone marrow. Cesium irradiators, however, have significant liabilities-including concerns around domestic security. Recently, X-ray irradiators have been implemented as a potential alternative to cesium sources. Only a small number of publications in the literature have attempted to compare these two modalities and, in most cases, the emphasis was on irradiation of human blood productions. We were able to find only a single study that directly compared X-ray and cesium technologies in the generation of murine bone marrow chimeras, a standard laboratory practice. This study focused on chimerism in the blood of recipient animals. In the present study, we begin by comparing cesium and X-ray based sources for irradiation, then transition to using X-ray-based systems for immunology models with an emphasis on immunotherapy of cancer in immunocompetent mouse models-specifically evaluating chimerism in the blood, spleen, and tumor microenvironment. While our data demonstrate that the two platforms are functionally comparable and suggest that X-ray based technology is a suitable alternative to cesium sources. We also highlight a difference in chimerism between the peripheral (blood, spleen) and tumor compartments that is observed using both technologies. While the overall degree of chimerism in the peripheral tissues is very high, the degree of chimerism in the tumor is cell type specific with T and NK cells showing lower chimerism than other cell types.
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Aortic stiffening is a major independent risk factor for cardiovascular diseases, cognitive dysfunction, and other chronic disorders of aging. Mitochondria-derived reactive oxygen species are a key source of arterial oxidative stress, which may contribute to arterial stiffening by promoting adverse structural changes-including collagen overabundance and elastin degradation-and enhancing inflammation, but the potential for mitochondria-targeted therapeutic strategies to ameliorate aortic stiffening with primary aging is unknown. We assessed aortic stiffness [pulse-wave velocity (aPWV)], ex vivo aortic intrinsic mechanical properties [elastic modulus (EM) of collagen and elastin regions], and aortic protein expression in young (~6 mo) and old (~27 mo) male C57BL/6 mice consuming normal drinking water (YC and OC) or water containing mitochondria-targeted antioxidant MitoQ (250 µM; YMQ and OMQ) for 4 wk. Both baseline and postintervention aPWV values were higher in OC vs. YC (post: 482 ± 21 vs. 420 ± 5 cm/s, P < 0.05). MitoQ had no effect in young mice but decreased aPWV in old mice (OMQ, 426 ± 20, P < 0.05 vs. OC). MitoQ did not affect age-associated increases in aortic collagen-region EM, collagen expression, or proinflammatory cytokine expression, but partially attenuated age-associated decreases in elastin region EM and elastin expression. Our results demonstrate that MitoQ reverses in vivo aortic stiffness in old mice and suggest that mitochondria-targeted antioxidants may represent a novel, promising therapeutic strategy for decreasing aortic stiffness with primary aging and, possibly, age-related clinical disorders in humans. The destiffening effects of MitoQ treatment may be at least partially mediated by attenuation/reversal of age-related aortic elastin degradation. NEW & NOTEWORTHY We show that 4 wk of treatment with the mitochondria-specific antioxidant MitoQ in mice completely reverses the age-associated elevation in aortic stiffness, assessed as aortic pulse-wave velocity. The destiffening effects of MitoQ treatment may be at least partially mediated by attenuation of age-related aortic elastin degradation. Our results suggest that mitochondria-targeted therapeutic strategies may hold promise for decreasing arterial stiffening with aging in humans, possibly decreasing the risk of many chronic age-related clinical disorders.
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Antioxidantes/farmacología , Aorta/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Rigidez Vascular/efectos de los fármacos , Animales , Aorta/metabolismo , Citocinas/metabolismo , Elastina/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Análisis de la Onda del Pulso/métodos , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismo , Vasodilatación/efectos de los fármacosRESUMEN
In the last 10-15 years, there has been a recognition that the catecholamines (norepinephrine, NE, and epinephrine, Epi) released by the sympathetic nervous system under stressful conditions promote tumor growth through a variety of mechanisms. Tumors recruit autonomic nerves during their development and NE is then released locally in the tumor microenvironment (TME). Acting through adrenergic receptors present on a variety of cells in the TME, NE and Epi induce proliferation, resistance to apoptosis, epithelial to mesenchymal transition, metastasis of tumor cells, angiogenesis, and inflammation in the TME. These pre-clinical studies have been conducted in mouse models whose care and housing parameters are outlined in "The Guide for the Care and Use of Laboratory Animals [1]. In particular, the Guide mandates that mice be housed at standardized sub-thermoneutral temperatures; however, this causes a state of chronic cold-stress and elevated levels of NE. Although mice are able to maintain a normal body temperature when kept at these cool temperatures, it is becoming clear that this cold-stress is sufficient to activate physiological changes which affect experimental outcomes. We find that when mice are housed under standard, sub-thermoneutral temperatures (~22 °C, ST), tumor growth is significantly greater than when mice are housed at thermoneutrality (~30 °C TT). We also find that the anti-tumor immune response is suppressed at ST and this immunosuppression can be reversed by housing mice at TT or by administration of propranolol (a ß-adrenergic receptor antagonist) to mice housed at ST. Furthermore, at ST tumors are more resistant to therapy and can also be sensitized to cytotoxic therapies by housing mice at TT or by treating mice with propranolol. The implications of these observations are particularly relevant to the way in which experiments conducted in preclinical models are interpreted and the findings implemented in the clinic. It may be that the disappointing failure of many new therapies to fulfill their promise in the clinic is related to an incomplete preclinical assessment in mouse models. Further, an expanded understanding of the efficacy of a therapy alone or in combination obtained by testing under a wider range of conditions would better predict how patients, who are under various levels of stress, might respond in a clinical setting. This may be particularly important to consider since we now appreciate that long term outcome of many therapies depends on eliciting an immune response.It is clear that the outcome of metabolic experiments, immunological investigations and therapeutic efficacy testing in tumors of mice housed at ST is restricted and expanding these experiments to include results obtained at TT may provide us with valuable information that would otherwise be overlooked.
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Neoplasias Experimentales , Animales , Enfermedad Crónica , Humanos , Ratones , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Neoplasias Experimentales/terapia , Sistema Nervioso/inmunología , Sistema Nervioso/patología , Estrés Psicológico/inmunología , Estrés Psicológico/patología , Estrés Psicológico/terapiaRESUMEN
The immune context of tumors has significant prognostic value and is predictive of responsiveness to several forms of therapy, including immunotherapy. We report here that CD8+ T-cell frequency and functional orientation within the tumor microenvironment is regulated by ß2-adrenergic receptor (ß-AR) signaling in host immune cells. We used three strategies-physiologic (manipulation of ambient thermal environment), pharmacologic (ß-blockers), and genetic (ß2-AR knockout mice) to reduce adrenergic stress signaling in two widely studied preclinical mouse tumor models. Reducing ß-AR signaling facilitated conversion of tumors to an immunologically active tumor microenvironment with increased intratumoral frequency of CD8+ T cells with an effector phenotype and decreased expression of programmed death receptor-1 (PD-1), in addition to an elevated effector CD8+ T-cell to CD4+ regulatory T-cell ratio (IFNγ+CD8+:Treg). Moreover, this conversion significantly increased the efficacy of anti-PD-1 checkpoint blockade. These data highlight the potential of adrenergic stress and norepinephrine-driven ß-AR signaling to regulate the immune status of the tumor microenvironment and support the strategic use of clinically available ß-blockers in patients to improve responses to immunotherapy. Cancer Res; 77(20); 5639-51. ©2017 AACR.
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Linfocitos T CD8-positivos/inmunología , Inmunoterapia/métodos , Neoplasias Mamarias Experimentales/inmunología , Melanoma Experimental/inmunología , Receptores Adrenérgicos beta 2/inmunología , Antagonistas de Receptores Adrenérgicos beta 2/farmacología , Animales , Femenino , Neoplasias Mamarias Experimentales/terapia , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Distribución Aleatoria , Transducción de Señal/inmunología , TemperaturaRESUMEN
BACKGROUND: Therapeutic resistance and tumor recurrence are two major hurdles in the treatment of pancreatic ductal adenocarcinoma. Recent findings suggest that both of these attributes are associated with a small subset of pancreatic tumor initiating cancer stem cells (CSCs). Here, we demonstrate that drozitumab, a human agonistic monoclonal antibody which binds the death receptor DR5, selectively eliminates CSCs, resulting in tumor growth inhibition and even regression of pancreatic tumors. METHODS: To examine the efficacy of drozitumab against pancreatic CSCs, we treated patient-derived pancreatic tumor xenografts (PDX) in immunocompromised SCID mice and evaluated tumor control. To assess apoptosis following drozitumab treatment, we identified the CSCs as CD24+, CD44+, and EpCAM+ by FACS analysis, and measured in vivo and in vitro levels of cleaved caspase-3. Lastly, in vitro evaluation of DR5 re-expression was performed using isolated patient pancreatic cancer xenograft cells along with the cell line, Panc-1. After treatment with drozitumab, the remaining DR5- cells were assessed by FACS analysis for DR5 expression at the cell surface at 8, 24 and 48 h post-treatment. All in vivo growth data was analyzed by 2-way Anova, incidence data was analyzed using Mantel-Cox, and in vitro studies statistics were performed with a t-test. RESULTS: We find that while 75-100 % of CSCs express DR5, only 25 % of bulk tumor cells express the death receptors at any one time. Consequently, drozitumab treatment of SCID mice bearing PDX kills higher percentages of CSCs than bulk tumor cells. Additionally, SCID mice implanted with isolated CSCs and then immediately treated with drozitumab fail to ever develop tumors. In vitro studies demonstrate that while drozitumab treatment reduces the DR5+ cell population, the remaining tumor cells begin to express DR5, suggesting a mechanism by which continuous administration of drozitumab can ultimately result in tumor regression despite the initially low percentage of DR5+ cells. CONCLUSIONS: Overall, our work reveals that treatment of pancreatic tumors with the drozitumab can lead to long-term tumor control by targeting both bulk cells and CSCs.
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Graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic cell transplantation, a potentially curative therapy for hematologic diseases. It has long been thought that murine bone marrow-derived T cells do not mediate severe GVHD because of their quantity and/or phenotype. During the course of experiments testing the impact of housing temperatures on GVHD, we discovered that this apparent resistance is a function of the relatively cool ambient housing temperature. Murine bone marrow-derived T cells have the ability to mediate severe GVHD in mice housed at a thermoneutral temperature. Specifically, mice housed at Institutional Animal Care and Use Committee-mandated, cool standard temperatures (â¼ 22°C) are more resistant to developing GVHD than are mice housed at thermoneutral temperatures (â¼ 30°C). We learned that the mechanism underlying this housing-dependent immunosuppression is associated with increased norepinephrine production and excessive signaling through ß-adrenergic receptor signaling, which is increased when mice are cold stressed. Treatment of mice housed at 22°C with a ß2-adrenergic antagonist reverses the norepinephrine-driven suppression of GVHD and yields similar disease to mice housed at 30°C. Conversely, administering a ß2-adrenergic agonist decreases GVHD in mice housed at 30°C. In further mechanistic studies using ß2-adrenergic receptor-deficient (ß2-AR(-/-)) mice, we found that it is host cell ß2-AR signaling that is essential for decreasing GVHD. These data reveal how baseline levels of ß-adrenergic receptor signaling can influence murine GVHD and point to the feasibility of manipulation of ß2-AR signaling to ameliorate GVHD in the clinical setting.
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Enfermedad Injerto contra Huésped/inmunología , Calor , Receptores Adrenérgicos beta 2/inmunología , Transducción de Señal/inmunología , Estrés Fisiológico/inmunología , Antagonistas de Receptores Adrenérgicos beta 2/farmacología , Animales , Femenino , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/terapia , Trasplante de Células Madre Hematopoyéticas , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptores Adrenérgicos beta 2/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Estrés Fisiológico/genéticaRESUMEN
We tested the hypothesis that aortic perivascular adipose tissue (PVAT) from young low-density lipoprotein receptor-deficient (LDLr(-/-)) mice promotes aortic stiffness and remodeling, which would be mediated by greater PVAT-derived IL-6 secretion. Arterial stiffness was assessed by aortic pulse wave velocity and with ex vivo intrinsic mechanical properties testing in young (4-6 mo old) wild-type (WT) and LDLr(-/-) chow-fed mice. Compared with WT mice, LDLr(-/-) mice had increased aortic pulse wave velocity (407 ± 18 vs. 353 ± 13 cm/s) and intrinsic mechanical stiffness (5,308 ± 623 vs. 3,355 ± 330 kPa) that was associated with greater aortic protein expression of collagen type I and advanced glycation end products (all P < 0.05 vs. WT mice). Aortic segments from LDLr(-/-) compared with WT mice cultured in the presence of PVAT had greater intrinsic mechanical stiffness (6,092 ± 480 vs. 3,710 ± 316 kPa), and this was reversed in LDLr(-/-) mouse arteries cultured without PVAT (3,473 ± 577 kPa, both P < 0.05). Collagen type I and advanced glycation end products were increased in LDLr(-/-) mouse arteries cultured with PVAT (P < 0.05 vs. WT mouse arteries), which was attenuated when arteries were cultured in the absence of PVAT (P < 0.05). PVAT from LDLr(-/-) mice secreted larger amounts of IL-6 (3.4 ± 0.1 vs. 2.3 ± 0.7 ng/ml, P < 0.05), and IL-6 neutralizing antibody decreased intrinsic mechanical stiffness in LDLr(-/-) aortic segments cultured with PVAT (P < 0.05). Collectively, these data provide evidence for a role of PVAT-derived IL-6 in the pathogenesis of aortic stiffness and remodeling in chow-fed LDLr(-/-) mice.
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Tejido Adiposo/metabolismo , Aorta/fisiopatología , Interleucina-6/metabolismo , Receptores de LDL/deficiencia , Rigidez Vascular , Animales , Aorta/metabolismo , Aorta/patología , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de la Onda del Pulso , Receptores de LDL/genéticaRESUMEN
Cancer research relies heavily on murine models for evaluating the anti-tumour efficacy of therapies. Here we show that the sensitivity of several pancreatic tumour models to cytotoxic therapies is significantly increased when mice are housed at a thermoneutral ambient temperature of 30 °C compared with the standard temperature of 22 °C. Further, we find that baseline levels of norepinephrine as well as the levels of several anti-apoptotic molecules are elevated in tumours from mice housed at 22 °C. The sensitivity of tumours to cytotoxic therapies is also enhanced by administering a ß-adrenergic receptor antagonist to mice housed at 22 °C. These data demonstrate that standard housing causes a degree of cold stress sufficient to impact the signalling pathways related to tumour-cell survival and affect the outcome of pre-clinical experiments. Furthermore, these data highlight the significant role of host physiological factors in regulating the sensitivity of tumours to therapy.
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Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pancreáticas/tratamiento farmacológico , Receptores Adrenérgicos beta 2/genética , Agonistas Adrenérgicos beta/farmacología , Albúminas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Cisplatino/farmacología , Humanos , Isoproterenol/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Paclitaxel/farmacología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transducción de Señal , Estrés Fisiológico , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Temperatura , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
The potential for immune cells to control cancers has been recognized for many decades, but only recently has real excitement begun to spread through the oncology community following clear evidence that therapeutic blockade of specific immune-suppressive mechanisms is enough to make a real difference in survival for patients with several different advanced cancers. However, impressive and encouraging as these new clinical data are, it is clear that more effort should be devoted toward understanding the full spectrum of factors within cancer patients, which have the potential to block or weaken antitumor activity by immune cells. The goal of this brief review is to highlight recent literature revealing interactive stress and metabolic pathways, particularly those mediated by the sympathetic nervous system, which may conspire to block immune cells from unleashing their full killing potential. There is exciting new information regarding the role of neurogenesis by tumors and adrenergic signaling in cancer progression (including metabolic changes associated with cachexia and lipolysis) and in regulation of immune cell function and differentiation. However, much more work is needed to fully understand how the systemic metabolic effects mediated by the brain and nervous system can be targeted for therapeutic efficacy in the setting of immunotherapy and other cancer therapies.
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Metabolismo Energético , Neoplasias/etiología , Neoplasias/metabolismo , Estrés Fisiológico , Estrés Psicológico , Animales , Progresión de la Enfermedad , Humanos , Tolerancia Inmunológica , Receptores Adrenérgicos/metabolismo , Transducción de SeñalRESUMEN
The production of new blood cells relies on a hierarchical network of hematopoietic stem and progenitor cells (HSPCs). To maintain lifelong hematopoiesis, HSPCs must be protected from ionizing radiation or other cytotoxic agents. For many years, murine models have been a valuable source of information regarding factors that either enhance or reduce the survival of HSPCs after exposure of marrow to ionizing radiation. In a recent series of studies, however, it has become clear that housing-related factors such as the cool room temperature required for laboratory mice can exert a surprising influence on the outcome of experiments. Here we report that the mild, but chronic cold-stress endured by mice housed under these conditions exerts a protective effect on HSPCs after both non-lethal and lethal doses of total body irradiation (TBI). Alleviation of this cold-stress by housing mice at a thermoneutral temperature (30°C) resulted in significantly greater baseline radiosensitivity to a lethal dose of TBI with more HSPCs from mice housed at thermoneutral temperature undergoing apoptosis following non-lethal TBI. Cold-stressed mice have elevated levels of norepinephrine, a key molecule of the sympathetic nervous system that binds to ß-adrenergic receptors. We show that blocking this signaling pathway in vivo through use of the ß-blocker propanolol completely mitigates the protective effect of cold-stress on HSPC apoptosis. Collectively this study demonstrates that chronic stress endured by the standard housing conditions of laboratory mice increases the resistance of HSPCs to TBI-induced apoptosis through a mechanism that depends upon ß-adrenergic signaling. Since ß-blockers are commonly prescribed to a wide variety of patients, this information could be important when predicting the clinical impact of HSPC sensitivity to TBI.
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Respuesta al Choque por Frío , Células Madre Hematopoyéticas/efectos de la radiación , Tolerancia a Radiación , Antagonistas Adrenérgicos beta/farmacología , Animales , Apoptosis , Células Cultivadas , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/fisiología , Vivienda para Animales , Ratones , Ratones Endogámicos C57BL , Propranolol/farmacología , Transducción de Señal , Irradiación Corporal TotalRESUMEN
PURPOSE: Researchers studying the murine response to stress generally use mice housed under standard, nationally mandated conditions as controls. Few investigators are concerned whether basic physical aspects of mouse housing could be an additional source of stress, capable of influencing the subsequent impact of an experimentally applied stressor. We have recently become aware of the potential for housing conditions to impact important physiological and immunological properties in mice. MATERIALS AND METHODS: Here we sought to determine whether housing mice at standard temperature (ST; 22 °C) vs. thermoneutral temperature (TT; 30 °C) influences baseline expression of heat shock proteins (HSPs) and their typical induction following a whole body heating. RESULTS: There were no significant differences in baseline expression of HSPs at ST and TT. However, in several cases, the induction of Hsp70, Hsp110 and Hsp90 in tissues of mice maintained at ST was greater than at TT following 6 h of heating (which elevated core body temperature to 39.5 °C). This loss of HSP induction was also seen when mice housed at ST were treated with propranolol, a ß-adrenergic receptor antagonist, used clinically to treat hypertension and stress. CONCLUSIONS: Taken together, these data show that housing temperature significantly influences the expression of HSPs in mice after whole body heating and thus should be considered when stress responses are studied in mice.
Asunto(s)
Temperatura Corporal/fisiología , Proteínas de Choque Térmico/metabolismo , Vivienda para Animales/normas , Hipertermia Inducida , Antagonistas Adrenérgicos beta/farmacología , Animales , Western Blotting/métodos , Respuesta al Choque por Frío/fisiología , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Ratones , Ratones Endogámicos BALB C , Norepinefrina/sangre , Propranolol/farmacología , Estrés Fisiológico/fisiologíaRESUMEN
Long conserved mechanisms maintain homeostasis in living creatures in response to a variety of stresses. However, continuous exposure to stress can result in unabated production of stress hormones, especially catecholamines, which can have detrimental health effects. While the long-term effects of chronic stress have well-known physiological consequences, recent discoveries have revealed that stress may affect therapeutic efficacy in cancer. Growing epidemiological evidence reveals strong correlations between progression-free and long-term survival and ß-blocker usage in cancer patients. In this review, we summarize the current understanding of how the catecholamines, epinephrine and norepinephrine, affect cancer cell survival and tumor progression. We also highlight new data exploring the potential contributions of stress to immunosuppression in the tumor microenvironment and the implications of these findings for the efficacy of immunotherapies.
Asunto(s)
Terapia de Inmunosupresión/métodos , Inmunosupresores/uso terapéutico , Neoplasias/patología , Neoplasias/psicología , Microambiente Tumoral , Animales , Ansiedad/complicaciones , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Epinefrina/metabolismo , Miedo , Homeostasis , Humanos , Tolerancia Inmunológica , Ratones , Norepinefrina/metabolismo , Receptores Adrenérgicos/metabolismo , Estrés Psicológico , Sistema Nervioso SimpáticoRESUMEN
Physiologically accurate mouse models of cancer are critical in the pre-clinical development of novel cancer therapies. However, current standardized animal-housing temperatures elicit chronic cold-associated stress in mice, which is further increased in the presence of tumor. This cold-stress significantly impacts experimental outcomes. Data from our lab and others suggest standard housing fundamentally alters murine physiology, and this can produce altered immune baselines in tumor and other disease models. Researchers may thus underestimate the efficacy of therapies that are benefitted by immune responses. A potential mediator, norepinephrine, also underlies stress pathways common in mice and humans. Therefore, research into mechanisms connecting cold-stress and norepinephrine signaling with immune depression in mice could highlight new combination therapies for humans to simultaneously target stress while stimulating anti-tumor immunity.
Asunto(s)
Respuesta al Choque por Frío/inmunología , Neoplasias Experimentales/inmunología , Neoplasias/inmunología , Aclimatación , Animales , Humanos , Tolerancia Inmunológica , Ratones Endogámicos , Neoplasias Experimentales/patología , Escape del TumorRESUMEN
We tested the hypothesis that superoxide signaling within aortic perivascular adipose tissue (PVAT) contributes to large elastic artery stiffening in old mice. Young (4-6 months), old (26-28 months), and old treated with 4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), a superoxide scavenger (1 mm in drinking water for 3 weeks), male C57BL6/N mice were studied. Compared with young, old had greater large artery stiffness assessed by aortic pulse wave velocity (aPWV, 436 ± 9 vs. 344 ± 5 cm s(-1)) and intrinsic mechanical testing (3821 ± 427 vs. 1925 ± 271 kPa) (both P < 0.05). TEMPOL treatment in old reversed both measures of arterial stiffness. Aortic PVAT superoxide production was greater in old (P < 0.05 vs. Y), which was normalized with TEMPOL. Compared with young, old controls had greater pro-inflammatory proteins in PVAT-conditioned media (P < 0.05). Young recipient mice transplanted with PVAT from old compared with young donors for 8 weeks had greater aPWV (409 ± 7 vs. 342 ± 8 cm s(-1)) and intrinsic mechanical properties (3197 ± 647 vs. 1889 ± 520 kPa) (both P < 0.05), which was abolished with TEMPOL supplementation in old donors. Tissue-cultured aortic segments from old in the presence of PVAT had greater mechanical stiffening compared with old cultured in the absence of PVAT and old with PVAT and TEMPOL (both, P < 0.05). In addition, PVAT-derived superoxide was associated with arterial wall hypertrophy and greater adventitial collagen I expression with aging that was attenuated by TEMPOL. Aging or TEMPOL treatment did not affect blood pressure. Our findings provide evidence for greater age-related superoxide production and pro-inflammatory proteins in PVAT, and directly link superoxide signaling in PVAT to large elastic artery stiffness.