RESUMEN
BACKGROUND: Metastatic breast cancer is a leading cause of cancer death in woman. Current treatment options are often associated with adverse side effects and poor outcomes, demonstrating the need for effective new treatments. Immunotherapies can provide durable outcomes in many cancers; however, limited success has been achieved in metastatic triple negative breast cancer. We tested whether combining different immunotherapies can target metastatic triple negative breast cancer in pre-clinical models. METHODS: Using primary and metastatic 4T1 triple negative mammary carcinoma models, we examined the therapeutic effects of oncolytic vesicular stomatitis virus (VSVΔM51) engineered to express reovirus-derived fusion associated small transmembrane proteins p14 (VSV-p14) or p15 (VSV-p15). These viruses were delivered alone or in combination with natural killer T (NKT) cell activation therapy mediated by adoptive transfer of α-galactosylceramide-loaded dendritic cells. RESULTS: Treatment of primary 4T1 tumors with VSV-p14 or VSV-p15 alone increased immunogenic tumor cell death, attenuated tumor growth, and enhanced immune cell infiltration and activation compared to control oncolytic virus (VSV-GFP) treatments and untreated mice. When combined with NKT cell activation therapy, oncolytic VSV-p14 and VSV-p15 reduced metastatic lung burden to undetectable levels in all mice and generated immune memory as evidenced by enhanced in vitro recall responses (tumor killing and cytokine production) and impaired tumor growth upon rechallenge. CONCLUSION: Combining NKT cell immunotherapy with enhanced oncolytic virotherapy increased anti-tumor immune targeting of lung metastasis and presents a promising treatment strategy for metastatic breast cancer.
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Células T Asesinas Naturales , Viroterapia Oncolítica , Virus Oncolíticos , Animales , Femenino , Ratones , Células T Asesinas Naturales/inmunología , Viroterapia Oncolítica/métodos , Humanos , Línea Celular Tumoral , Virus Oncolíticos/genética , Virus Oncolíticos/inmunología , Inmunoterapia/métodos , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/inmunología , Neoplasias de la Mama Triple Negativas/terapia , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patología , Terapia Combinada , Metástasis de la Neoplasia , Vesiculovirus/genética , Células Dendríticas/inmunología , Neoplasias de la Mama/terapia , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Modelos Animales de EnfermedadRESUMEN
Despite numerous therapeutic options, multidrug resistance (MDR) remains an obstacle to successful breast cancer therapy. Jadomycin B, a natural product derived from Streptomyces venezuelae ISP5230, maintains cytotoxicity in MDR human breast cancer cells. Our objectives were to evaluate the pharmacokinetics, toxicity, anti-tumoral, and anti-metastatic effects of jadomycin B in zebrafish larvae and mice. In a zebrafish larval xenograft model, jadomycin B significantly reduced the proliferation of human MDA-MB-231 cells at or below its maximum tolerated dose (40 µm). In female Balb/C mice, a single intraperitoneal dose (6 mg/kg) was rapidly absorbed with a maximum serum concentration of 3.4 ± 0.27 µm. Jadomycin B concentrations declined biphasically with an elimination half-life of 1.7 ± 0.058 h. In the 4T1 mouse mammary carcinoma model, jadomycin B (12 mg/kg every 12 h from day 6 to 15 after tumor cell injection) decreased primary tumor volume compared to vehicle control. Jadomycin B-treated mice did not exhibit weight loss, nor significant increases in biomarkers of impaired hepatic (alanine aminotransferase) and renal (creatinine) function. In conclusion, jadomycin B demonstrated a good safety profile and provided partial anti-tumoral effects, warranting further dose-escalation safety and efficacy studies in MDR breast cancer models.
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Neoplasias de la Mama , Pez Cebra , Humanos , Femenino , Animales , Ratones , Proyectos Piloto , XenoinjertosRESUMEN
BACKGROUND: Pancreatic cancer is one of the leading causes of cancer death, with a 5-year -year survival rate of less than 10%. This results from late detection, high rates of metastasis, and resistance to standard chemotherapies. Furthermore, chemotherapy and radiation are associated with significant morbidity, underscoring the need for novel therapies. Recent clinical studies have shown that immunotherapies can provide durable outcomes in cancer patients, but successes in pancreatic cancer have been limited. It is likely that novel and combined therapies will be needed to achieve clinical benefits. METHODS: Using experimental mouse models of pancreatic ductal adenocarcinoma, we examined natural killer T (NKT) cell activation therapy in combination with a recombinant oncolytic vesicular stomatitis virus (VSVΔM51) engineered to express the cytokine IL-15 (VSV-IL-15). Panc02 pancreatic ductal carcinoma cells were implanted subcutaneously or orthotopically into syngeneic C57BL/6 mice. Mice were then treated with VSV expressing green fluorescent protein (VSV-GFP) or VSV-IL-15 and/or NKT cell activation therapy via delivery of α-GalCer-loaded DCs. We further assessed whether the addition of PD-1 blockade could increase the therapeutic benefit of our combination treatment. Three days after NKT cell activation, some groups of mice were treated with anti-PD-1 antibodies weekly for 3 weeks. RESULTS: VSV-GFP and VSV-IL-15 mediated equal killing of human and mouse pancreatic cancer lines in vitro. In vivo, VSV-IL-15 combined with NKT cell activation therapy to enhance tumor regression and increase survival time over individual treatments, and was also superior to NKT cell therapy combined with VSV-GFP. Enhanced tumor control was associated with increased immune cell infiltration and anti-tumor effector functions (cytotoxicity and cytokine production). While ineffective as a monotherapy, the addition of blocking PD-1 antibodies to the combined protocol sustained immune cell activation and effector functions, resulting in prolonged tumor regression and complete tumor clearance in 20% of mice. Mice who cleared the initial tumor challenge exhibited reduced tumor growth uponon rechallenge, consistent with the formation of immune memory. CONCLUSION: TThese results demonstrate that NKT cell immunotherapy combined with oncolytic VSV-IL-15 virotherapy and PD-1 blockade enhances tumor control and presents a promising treatment strategy for targeting pancreatic cancer.
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Carcinoma Ductal Pancreático , Células T Asesinas Naturales , Viroterapia Oncolítica , Virus Oncolíticos , Neoplasias Pancreáticas , Animales , Carcinoma Ductal Pancreático/terapia , Línea Celular Tumoral , Citocinas/metabolismo , Humanos , Inmunoterapia , Interleucina-15/genética , Interleucina-15/metabolismo , Ratones , Ratones Endogámicos C57BL , Viroterapia Oncolítica/métodos , Neoplasias Pancreáticas/terapia , Receptor de Muerte Celular Programada 1/metabolismo , Virus de la Estomatitis Vesicular Indiana/metabolismo , Vesiculovirus , Neoplasias PancreáticasRESUMEN
Immunomodulation of mast cell (MC) activity is warranted in allergic and inflammatory diseases where MCs have a central role in pathogenesis. Targeting Siglec-8, an inhibitory receptor on MCs and eosinophils, has shown promising activity in preclinical and clinical studies. While the intracellular pathways that regulate Siglec-8 activity in eosinophils have been well studied, the signaling mechanisms that lead to MC inhibition have not been fully elucidated. Here, we evaluate the intracellular signaling pathways of Siglec-8-mediated inhibition in primary MCs using an anti-Siglec-8 monoclonal antibody (mAb). Phospho-proteomic profiling of FcεRI-activated MCs revealed Siglec-8 mAb-treatment globally inhibited proximal and downstream kinases, leading to attenuated MC activation and degranulation. In fact, Siglec-8 was found to directly interact with FcεRI signaling molecules. Siglec-8 inhibition was dependent on both cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that interact with the SH2 containing protein phosphatase Shp-2 upon Siglec-8 phosphorylation. Taken together, these data support a model in which Siglec-8 regulates proximal FcεRI-induced phosphorylation events through phosphatase recruitment and interaction with FcεRIγ, resulting in global inhibition of MCs upon Siglec-8 mAb engagement.
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Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Lectinas/metabolismo , Mastocitos/inmunología , Receptores de IgE/metabolismo , Animales , Degranulación de la Célula , Humanos , Ratones , Ratones Endogámicos C57BL , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteómica , Transducción de SeñalRESUMEN
BACKGROUND: Oncolytic viruses reduce tumor burden in animal models and have generated promising results in clinical trials. However, it is likely that oncolytic viruses will be more effective when used in combination with other therapies. Current therapeutic approaches, including chemotherapeutics, come with dose-limiting toxicities. Another option is to combine oncolytic viruses with immunotherapeutic approaches. METHODS: Using experimental models of metastatic 4T1 breast cancer and ID8 ovarian peritoneal carcinomatosis, we examined natural killer T (NKT) cell-based immunotherapy in combination with recombinant oncolytic vesicular stomatitis virus (VSV) or reovirus. 4T1 mammary carcinoma cells or ID8 ovarian cancer cells were injected into syngeneic mice. Tumor-bearing mice were treated with VSV or reovirus followed by activation of NKT cells via the intravenous administration of autologous dendritic cells loaded with the glycolipid antigen α-galactosylceramide. The effects of VSV and reovirus on immunogenic cell death (ICD), cell viability and immunogenicity were tested in vitro. RESULTS: VSV or reovirus treatments followed by NKT cell activation mediated greater survival in the ID8 model than individual therapies. The regimen was less effective when the treatment order was reversed, delivering virus treatments after NKT cell activation. In the 4T1 model, VSV combined with NKT cell activation increased overall survival and decreased metastatic burden better than individual treatments. In contrast, reovirus was not effective on its own or in combination with NKT cell activation. In vitro, VSV killed a panel of tumor lines better than reovirus. VSV infection also elicited greater increases in mRNA transcripts for proinflammatory cytokines, chemokines, and antigen presentation machinery compared with reovirus. Oncolytic VSV also induced the key hallmarks of ICD (calreticulin mobilization, plus release of ATP and HMGB1), while reovirus only mobilized calreticulin. CONCLUSION: Taken together, these results demonstrate that oncolytic VSV and NKT cell immunotherapy can be effectively combined to decrease tumor burden in models of metastatic breast and ovarian cancers. Oncolytic VSV and reovirus induced differential responses in our models which may relate to differences in virus activity or tumor susceptibility.
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Neoplasias de la Mama/terapia , Inmunoterapia Adoptiva , Células T Asesinas Naturales/trasplante , Viroterapia Oncolítica , Virus Oncolíticos/inmunología , Neoplasias Ováricas/terapia , Neoplasias Peritoneales/terapia , Reoviridae/inmunología , Vesiculovirus/inmunología , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Neoplasias de la Mama/virología , Línea Celular Tumoral , Chlorocebus aethiops , Terapia Combinada , Citocinas/metabolismo , Citotoxicidad Inmunológica , Femenino , Interacciones Huésped-Patógeno , Activación de Linfocitos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células T Asesinas Naturales/inmunología , Virus Oncolíticos/patogenicidad , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Neoplasias Ováricas/virología , Neoplasias Peritoneales/inmunología , Neoplasias Peritoneales/secundario , Neoplasias Peritoneales/virología , Reoviridae/patogenicidad , Células Vero , Vesiculovirus/patogenicidadRESUMEN
Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection represents a global health crisis. Immune cell activation via pattern recognition receptors has been implicated as a driver of the hyperinflammatory response seen in COVID-19. However, our understanding of the specific immune responses to SARS-CoV-2 remains limited. Mast cells (MCs) and eosinophils are innate immune cells that play pathogenic roles in many inflammatory responses. Here we report MC-derived proteases and eosinophil-associated mediators are elevated in COVID-19 patient sera and lung tissues. Stimulation of viral-sensing toll-like receptors in vitro and administration of synthetic viral RNA in vivo induced features of hyperinflammation, including cytokine elevation, immune cell airway infiltration, and MC-protease production-effects suppressed by an anti-Siglec-8 monoclonal antibody which selectively inhibits MCs and depletes eosinophils. Similarly, anti-Siglec-8 treatment reduced disease severity and airway inflammation in a respiratory viral infection model. These results suggest that MC and eosinophil activation are associated with COVID-19 inflammation and anti-Siglec-8 antibodies are a potential therapeutic approach for attenuating excessive inflammation during viral infections.
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Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , COVID-19/inmunología , Eosinófilos/inmunología , Lectinas/inmunología , Mastocitos/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/inmunología , SARS-CoV-2/inmunología , Receptores Toll-Like/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/metabolismo , COVID-19/metabolismo , COVID-19/prevención & control , COVID-19/virología , Estudios de Casos y Controles , Citocinas/metabolismo , Modelos Animales de Enfermedad , Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Eosinófilos/virología , Interacciones Huésped-Patógeno , Humanos , Lectinas/antagonistas & inhibidores , Lectinas/genética , Lectinas/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Mastocitos/virología , Ratones Transgénicos , Péptido Hidrolasas/metabolismo , Infecciones por Virus Sincitial Respiratorio/metabolismo , Infecciones por Virus Sincitial Respiratorio/prevención & control , Infecciones por Virus Sincitial Respiratorio/virología , Receptores Toll-Like/metabolismoRESUMEN
In addition to their well characterized role in mediating IgE-dependent allergic diseases, aberrant accumulation and activation of mast cells (MCs) is associated with many non-allergic inflammatory diseases, whereby their activation is likely triggered by non-IgE stimuli (e.g., IL-33). Siglec-8 is an inhibitory receptor expressed on MCs and eosinophils that has been shown to inhibit IgE-mediated MC responses and reduce allergic inflammation upon ligation with a monoclonal antibody (mAb). Herein, we evaluated the effects of an anti-Siglec-8 mAb (anti-S8) in non-allergic disease models of experimental cigarette-smoke-induced chronic obstructive pulmonary disease and bleomycin-induced lung injury in Siglec-8 transgenic mice. Therapeutic treatment with anti-S8 inhibited MC activation and reduced recruitment of immune cells, airway inflammation, and lung fibrosis. Similarly, using a model of MC-dependent, IL-33-induced inflammation, anti-S8 treatment suppressed neutrophil influx, and cytokine production through MC inhibition. Transcriptomic profiling of MCs further demonstrated anti-S8-mediated downregulation of MC signaling pathways induced by IL-33, including TNF signaling via NF-κB. Collectively, these findings demonstrate that ligating Siglec-8 with an antibody reduces non-allergic inflammation and inhibits IgE-independent MC activation, supporting the evaluation of an anti-Siglec-8 mAb as a therapeutic approach in both allergic and non-allergic inflammatory diseases in which MCs play a role.
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Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Lectinas/metabolismo , Mastocitos/inmunología , Neumonía/inmunología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Sistema Respiratorio/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos B/genética , Degranulación de la Célula , Fumar Cigarrillos , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Inmunoglobulina E/metabolismo , Interleucina-33/metabolismo , Lectinas/genética , Ratones , Ratones Transgénicos , FN-kappa B/metabolismo , Activación Neutrófila , Transducción de SeñalRESUMEN
Triple-negative breast cancer (TNBC) is an invasive subtype of breast cancer but paradoxically associated with increased tumor-infiltrating leukocytes. The molecular and cellular mechanisms underlying TNBC immunobiology are incompletely understood. Interleukin (IL)-17A is a pro-inflammatory cytokine that has both pro- and anti-tumor effects and found in 40-80% of TNBC samples. We report here that IL-17A mRNA and protein are detectable in some human TNBC cell lines and further upregulated by IL-23 and LPS stimulation. Furthermore, the impact of tumor-derived IL-17A in host immune response and tumor growth was examined using murine TNBC 4T1 mammary carcinoma cells transduced with an adenoviral vector expressing IL-17A (AdIL-17A) or control vector (Addl). Compared to Addl-transduction, AdIL-17A-transduction enhanced 4T1 tumor growth and lung metastasis in vivo, which was associated with a marked expansion of myeloid-derived suppressor cells (MDSCs). However, AdIL-17A-transduction also induced strong organ-specific and time-dependent immune activation indicated by dynamic changes of NK cells, B cells, CD4, and CD8 T cells in peripheral blood, lung, and tumor site, as well as the plasma levels of IFNγ. Such findings highlight that tumor-associated IL-17A induces concurrent immune activation and immune suppression. Administration of anti-Gr1 or anti-G-CSF antibody effectively depleted MDSCs in vivo, markedly reducing the growth of AdIL-17A-transduced 4T1 tumors, and eliminating lung metastasis. Collectively, our study demonstrates that MDSC depletion is an effective and practical approach for treating IL-17A-enriched mammary carcinomas.
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Neoplasias de la Mama/metabolismo , Interleucina-17/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Células Supresoras de Origen Mieloide/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Proliferación Celular/fisiología , Femenino , Humanos , Células Asesinas Naturales/metabolismo , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos BALB C , Células Mieloides/metabolismoRESUMEN
(1) Background: The cannabinoid 2 receptor (CB2R) is a promising anti-inflammatory drug target and development of selective CB2R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB2R ligands: CB2R agonists, RO6871304, and RO6871085, as well as a CB2R inverse agonist, RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB2R interactions, binding, cell signaling (ß-arrestin and cAMP) and early absorption, distribution, metabolism, excretion, and toxicology (ADMET) profiling of these receptor ligands. All ligands were evaluated for their efficacy to modulate leukocyte-neutrophil activity, in comparison to the reported CB2R ligand, HU910, using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB2R-/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB2R-/- mice, and in vivo in WT mice with EIU using adoptive transfer of WT and CB2R-/- neutrophils, respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB2R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB2R and selectivity for CB2R > CB1R, with both ligands acting as full agonists in cAMP and ß-arrestin assays (EC50s in low nM range). When tested in EIU, topical application of RO6871304 and RO6871085 decreased leukocyte-endothelial adhesion and this effect was antagonized by the inverse agonist, RO6851228. The CB2R agonist, RO6871304, decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB2R-/-, and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB2R and have good immunomodulating actions in the eye. RO6871304 and RO6871085, as well as HU910, decreased leukocyte adhesion in EIU through inhibition of resident ocular immune cells. The data generated with these three structurally-diverse and highly-selective CB2R agonists support selective targeting of CB2R for treating ocular inflammatory diseases.
Asunto(s)
Antiinflamatorios/administración & dosificación , Agonistas de Receptores de Cannabinoides/administración & dosificación , Endotoxinas/efectos adversos , Receptor Cannabinoide CB2/antagonistas & inhibidores , Uveítis/tratamiento farmacológico , Animales , Antiinflamatorios/química , Antiinflamatorios/farmacología , Agonistas de Receptores de Cannabinoides/química , Agonistas de Receptores de Cannabinoides/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Masculino , Ratones , Ratones Noqueados , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Receptor Cannabinoide CB2/química , Receptor Cannabinoide CB2/genética , Transducción de Señal , Uveítis/inducido químicamente , Uveítis/inmunologíaRESUMEN
BACKGROUND: Cell-assisted lipotransfer involves enrichment of autologous fat with supraphysiologic numbers of adipose-derived stem cells to improve graft take. Adipose-derived stem cells have been shown to promote cancer progression, raising concerns over the safety of adipose-derived stem cells and cell-assisted lipotransfer in postoncologic breast reconstruction. The authors compared the effect of adipose-derived stem cells alone, cell-assisted lipotransfer, and conventional fat grafting on breast cancer growth and metastasis. METHODS: Proliferation and migration of murine 4T1 breast cancer cells cultured in control medium or mouse adipose-derived stem cell- or fat graft-conditioned medium were assessed by flow cytometry and scratch assay, respectively. Transcription levels of arginase-1, transforming growth factor-ß, and vascular endothelial growth factor were assessed in adipose-derived stem cells and fat graft by quantitative reverse transcription polymerase chain reaction. An orthotopic mouse tumor model was used to evaluate breast cancer progression and metastasis. 4T1 cells were injected into the mammary pad of female BALB/c mice. Six days later, tumors were injected with saline, adipose-derived stem cells, fat graft, or cell-assisted lipotransfer (n = 7 per group). Two weeks later, primary tumors were examined by immunohistochemistry and lung metastasis was quantified. RESULTS: Adipose-derived stem cell-conditioned medium increased cancer cell proliferation (p = 0.03); migration (p < 0.01); and transcription of arginase-1, transforming growth factor-ß, and vascular endothelial growth factor compared to fat graft-conditioned or control medium (p < 0.02). Tumor-site injection with adipose-derived stem cells alone led to increased primary tumor growth and lung metastasis compared to control, fat graft, or cell-assisted lipotransfer groups (p < 0.05). Adipose-derived stem cell injection increased CD31 vascular density in tumors (p < 0.01). CONCLUSION: Adipose-derived stem cells alone, but not conventional fat graft or cell-assisted lipotransfer, promote breast cancer cell proliferation and invasiveness in vitro and in vivo.
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Adipocitos/fisiología , Tejido Adiposo/trasplante , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Neoplasias Mamarias Animales/patología , Adipocitos/citología , Animales , Autoinjertos , Medios de Cultivo Condicionados , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB C , Distribución Aleatoria , Estadísticas no Paramétricas , Células Madre/citología , Células Madre/fisiología , Células Tumorales CultivadasRESUMEN
Activated platelets promote the proliferation and metastatic potential of cancer cells. Platelet activation is largely mediated through ADP engagement of purinergic P2Y12 receptors on platelets. We examined the potential of the reversible P2Y12 inhibitor ticagrelor, an agent used clinically to prevent cardiovascular and cerebrovascular events, to reduce tumor growth and metastasis. In vitro, MCF-7, MDA-MB-468, and MDA-MB-231 human mammary carcinoma cells exhibited decreased interaction with platelets treated with ticagrelor compared to untreated platelets. Prevention of tumor cell-platelet interactions through pretreatment of platelets with ticagrelor did not improve natural killer cell-mediated tumor cell killing of K562 myelogenous leukemia target cells. Additionally, ticagrelor had no effect on proliferation of 4T1 mouse mammary carcinoma cells co-cultured with platelets, or on primary 4T1 tumor growth. In an orthotopic 4T1 breast cancer model, ticagrelor (10 mg/kg), but not clopidogrel (10 mg/kg) or saline, resulted in reduced metastasis and improved survival. Ticagrelor treatment was associated with a marked reduction in tumor cell-platelet aggregates in the lungs at 10, 30 and 60 min post-intravenous inoculation. These findings suggest a role for P2Y12-mediated platelet activation in promoting metastasis, and provide support for the use of ticagrelor in the prevention of breast cancer spread.
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Plaquetas/efectos de los fármacos , Neoplasias de la Mama/patología , Neoplasias Mamarias Experimentales/patología , Inhibidores de Agregación Plaquetaria/farmacología , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y12/efectos de los fármacos , Ticagrelor/farmacología , Animales , Plaquetas/inmunología , Plaquetas/metabolismo , Neoplasias de la Mama/inmunología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Células Asesinas Naturales/inmunología , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/inmunología , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Selectina-P/metabolismo , Activación Plaquetaria/fisiología , Receptores Purinérgicos P2Y12/fisiología , Tasa de SupervivenciaRESUMEN
Natural killer T (NKT) cells are glycolipid-reactive lymphocytes that promote cancer control. In previous studies, NKT-cell activation improved survival and antitumor immunity in a postsurgical mouse model of metastatic breast cancer. Herein, we investigated whether NKT-cell activation could be combined with chemotherapeutic agents to augment therapeutic outcomes. Gemcitabine and cyclophosphamide analogues enhanced the potential immunogenicity of 4T1 mammary carcinoma cells by increasing the expression of antigen-presenting molecules (MHC-I, MHC-II, and CD1d) and promoting exposure or release of immunogenic cell death markers (calreticulin, HMGB1, and ATP). In 4T1 primary tumor and postsurgical metastasis models, BALB/c mice were treated with cyclophosphamide or gemcitabine. NKT cells were then activated by transfer of dendritic cells loaded with the glycolipid antigen α-galactosylceramide (α-GalCer). Chemotherapeutic treatments did not impact NKT-cell activation but enhanced recruitment into primary tumors. Cyclophosphamide, gemcitabine, or α-GalCer-loaded dendritic cell monotherapies decreased tumor growth in the primary tumor model and reduced metastatic burden and prolonged survival in the metastasis model. Combining chemotherapeutics with NKT-cell activation therapy significantly enhanced survival, with surviving mice exhibiting attenuated tumor growth following a second tumor challenge. The frequency of myeloid-derived suppressor cells was reduced by gemcitabine, cyclophosphamide, or α-GalCer-loaded dendritic cell treatments; cyclophosphamide also reduced the frequency of regulatory T cells. Individual treatments increased immune cell activation, cytokine polarization, and cytotoxic responses, although these readouts were not enhanced further by combining therapies. These findings demonstrate that NKT-cell activation therapy can be combined with gemcitabine or cyclophosphamide to target tumor burden and enhance protection against tumor recurrence. Cancer Immunol Res; 5(12); 1086-97. ©2017 AACR.
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Antineoplásicos/farmacología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Inmunoterapia , Células T Asesinas Naturales/inmunología , Animales , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/terapia , Muerte Celular/efectos de los fármacos , Muerte Celular/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Terapia Combinada , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunomodulación/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Células T Asesinas Naturales/metabolismoRESUMEN
The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral fusogens (â¼100-150 amino acids) and efficiently induce cell-cell fusion and syncytium formation in multiple cell types. Syncytium formation enhances cell-cell virus transmission and may also induce immunogenic cell death, a form of apoptosis that stimulates immune recognition of tumor cells. These properties suggest that FAST proteins might serve to enhance oncolytic virotherapy. The oncolytic activity of recombinant VSVΔM51 (an interferon-sensitive vesicular stomatitis virus [VSV] mutant) encoding the p14 FAST protein (VSV-p14) was compared with a similar construct encoding GFP (VSV-GFP) in cell culture and syngeneic BALB/c tumor models. Compared with VSV-GFP, VSV-p14 exhibited increased oncolytic activity against MCF-7 and 4T1 breast cancer spheroids in culture and reduced primary 4T1 breast tumor growth in vivo. VSV-p14 prolonged survival in both primary and metastatic 4T1 breast cancer models, and in a CT26 metastatic colon cancer model. As with VSV-GFP, VSV-p14 preferentially replicated in vivo in tumors and was cleared rapidly from other sites. Furthermore, VSV-p14 increased the numbers of activated splenic CD4, CD8, natural killer (NK), and natural killer T (NKT) cells, and increased the number of activated CD4 and CD8 cells in tumors. FAST proteins may therefore provide a multi-pronged approach to improving oncolytic virotherapy via syncytium formation and enhanced immune stimulation.
RESUMEN
Proliferative vitreoretinopathy (PVR) can develop after ocular trauma or inflammation and is a common complication of surgery to correct retinal detachment. Currently, there are no pharmacological treatments for PVR. Cannabinoids acting at cannabinoid 2 receptor (CB2R) can decrease inflammation and fibrosis. The objective of this study was to examine the anti-inflammatory actions of CB2R as a candidate novel therapeutic target in experimental PVR. PVR was induced by intravitreal injection of dispase in wild-type (WT) and CB2R genetic knockout (CB2R-/-) mice. Ocular pathology was studied at 24 h or one week after dispase injection. CB2R modulation was examined in WT mice, using the CB2R agonist, HU308, and the CB2R antagonist, AM630. Histopathological scoring and quantification of microglia was used to evaluate tissue pathology. Quantitative PCR and multiplex assays were used to assess changes in proinflammatory cytokines. Intravital microscopy (IVM) was used to visualize and quantify leukocyte-endothelial adhesion to the iridial microcirculation. Activation of CB2R with HU308 in WT mice with PVR decreased mean histopathological scores, the number of microglia, and leukocyte adhesion compared to vehicle-treated animals. Conversely, an increase in histopathological scores and activated microglia was observed in PVR animals after treatment with AM630. CB2R-/- mice with PVR exhibited exacerbated ocular histopathology, increased microglia numbers, and elevated protein levels of cytokines as compared to WT mice. In conclusion, our results indicate that intervention at early stage PVR with CB2R agonists reduces ocular inflammation and disease severity. CB2R may represent a therapeutic target to prevent PVR progression and vision loss. This article is part of the Special Issue entitled 'Lipid Sensing G Protein-Coupled Receptors in the CNS'.
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Antiinflamatorios no Esteroideos/farmacología , Moduladores de Receptores de Cannabinoides/farmacología , Receptor Cannabinoide CB2/metabolismo , Vitreorretinopatía Proliferativa/tratamiento farmacológico , Vitreorretinopatía Proliferativa/inmunología , Animales , Cannabinoides/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Endopeptidasas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Células Endoteliales/patología , Indoles/farmacología , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Leucocitos/patología , Lipopolisacáridos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/efectos de los fármacos , Microglía/inmunología , Microglía/patología , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/antagonistas & inhibidores , Receptor Cannabinoide CB2/genética , Retina/efectos de los fármacos , Retina/inmunología , Retina/patología , Vitreorretinopatía Proliferativa/patologíaRESUMEN
Crosstalk interactions between dendritic cells (DCs) and invariant natural killer T (iNKT) cells are important in regulating antitumor responses elicited by glycolipid antigens. iNKT cells constitutively express the chemokine receptor CXCR6, while cytokine-activated DCs upregulate the transmembrane chemokine ligand, CXCL16. This study examined the co-stimulatory role of CXCR6/CXCL16 interactions in glycolipid-dependent iNKT cell activation and tumor control. Spleen and liver DCs in wild-type mice, but not iNKT cell deficient (Jα18(-/-)) mice, transiently upregulated surface CXCL16 following in vivo administration of the glycolipid antigen α-galactosylceramide. Recombinant CXCL16 did not directly induce iNKT cell activation in vitro but enhanced interferon (IFN)-γ production when mouse or human iNKT cells were stimulated with plate-bound anti-CD3. Compared with glycolipid-loaded CXCL16(neg) DCs, CXCL16(hi) DCs induced higher levels of IFNγ production in iNKT cell cultures and following adoptive transfer in vivo. The number of IFNγ(+) iNKT cells and expansion of T-bet(+) iNKT cells were reduced in vivo when CXCL16(-/-) DCs were used to activate iNKT cells. Enhanced IFNγ production in vivo was not dependent on CXCR6 expression on natural killer (NK) cells. Adoptive transfer of glycolipid-loaded CXCL16(hi) DCs provided superior protection against tumor metastasis compared to CXCL16(neg) DC transfers. Similarly, wild-type DCs provided superior protection against metastasis compared with CXCL16(-/-) DCs. These experiments implicate an important role for CXCR6/CXCL16 interactions in regulating iNKT cell IFNγ production and tumor control. The selective use of CXCL16(hi) DCs in adoptive transfer immunotherapies may prove useful for enhancing T helper (Th) type 1 responses and clinical outcomes in cancer patients.
RESUMEN
Natural killer T (NKT) cells are glycolipid-reactive T lymphocytes that function in immunosurveillance and immune regulation. However, reduced tumor control in NKT cell-deficient Jα18(-/-) mice may be confounded by an overall reduction in T-cell receptor (TCR) repertoire diversity in these animals. Mechanistic studies are also hindered by a lack of tools to target molecules specifically in NKT cells. To address these issues, we developed protocols to expand functional NKT cells and stably reconstitute them in Jα18(-/-) mice. In vivo delivery of α-galactosylceramide (α-GalCer)-loaded dendritic cells expanded NKT cells in wild-type mice without skewing CD4 or TCR Vß expression profiles. Expanded NKT cells exhibited enhanced cytokine responses upon re-stimulation with glycolipid or CD3 ligation. Adoptive transfer of recently expanded wild-type or interferon (IFN)-γ(-/-) NKT cells protected recipient Jα18(-/-) mice from B16 melanoma metastasis without the need for additional glycolipid stimulation. However, NKT cell reconstitution in recipient Jα18(-/-) mice was short lived. Long-term reconstitution was only achieved when expanded NKT cells were transferred into sublethally irradiated recipients. Thirty days after transfer, NKT cell numbers, phenotype and α-GalCer-induced cytokine responses were equivalent to naive wild-type mice. Jα18(-/-) recipients reconstituted with wild-type or IFN-γ(-/-) NKT cells were both protected from B16 melanoma metastasis following α-GalCer treatment, and NK cell transactivation was intact in mice reconstituted with IFN-γ(-/-) NKT cells. These studies validate the use of reconstitution protocols to investigate the mechanisms of NKT cell immune function, demonstrating that NKT cell-derived IFN-γ and the altered TCR repertoire in Jα18(-/-) mice do not impact NKT cell-mediated antitumor responses.
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Melanoma Experimental/inmunología , Melanoma Experimental/patología , Modelos Inmunológicos , Células T Asesinas Naturales/inmunología , Animales , Biomarcadores/metabolismo , Proliferación Celular , Células Dendríticas/inmunología , Galactosilceramidas , Interferón gamma/deficiencia , Interferón gamma/metabolismo , Activación de Linfocitos/inmunología , Ratones Endogámicos C57BL , Fenotipo , Activación TranscripcionalRESUMEN
Chemotherapy has historically been thought to induce cancer cell death in an immunogenically silent manner. However, recent studies have demonstrated that therapeutic outcomes with specific chemotherapeutic agents (e.g. anthracyclines) correlate strongly with their ability to induce a process of immunogenic cell death (ICD) in cancer cells. This process generates a series of signals that stimulate the immune system to recognize and clear tumor cells. Extensive studies have revealed that chemotherapy-induced ICD occurs via the exposure/release of calreticulin (CALR), ATP, chemokine (C-X-C motif) ligand 10 (CXCL10) and high mobility group box 1 (HMGB1). This review provides an in-depth look into the concepts and mechanisms underlying CALR exposure, activation of the Toll-like receptor 3/IFN/CXCL10 axis, and the release of ATP and HMGB1 from dying cancer cells. Factors that influence the impact of ICD in clinical studies and the design of therapies combining chemotherapy with immunotherapy are also discussed.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Inmunoterapia/métodos , Neoplasias/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Calbindina 2/metabolismo , Muerte Celular/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Proteína HMGB1/metabolismo , Humanos , Interferón Tipo I/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Receptor Toll-Like 3/metabolismo , Escape del TumorRESUMEN
BACKGROUND: Autologous fat grafting is a popular reconstructive technique, but is limited by inconsistent graft retention. The authors examined whether a widely available, clinically safe antioxidant, N-acetylcysteine, could improve adipose-derived stem cell survival and graft take when added to tumescent solution during fat harvest. METHODS: Inguinal fat pads were harvested from C57BL/6 mice using tumescent solution with or without N-acetylcysteine. Flow cytometric, proliferation, and differentiation assays were performed on isolated primary adipose-derived stem cells and 3T3-L1 preadipocytes treated with or without hydrogen peroxide and/or N-acetylcysteine. N-Acetylcysteine-treated or control grafts were injected under recipient mouse scalps and assessed by serial micro-computed tomographic volumetric analysis. Explanted grafts underwent immunohistochemical analysis. RESULTS: In culture, N-acetylcysteine protected adipose-derived stem cells from oxidative stress and improved cell survival following hydrogen peroxide treatment. Combined exposure to both N-acetylcysteine and hydrogen peroxide led to a 200-fold increase in adipose-derived stem cell proliferation, significantly higher than with either agent alone. N-Acetylcysteine decreased differentiation of adipose-derived stem cells into mature adipocytes, as evidenced by decreased transcription of adipocyte differentiation markers and reduced Oil Red-O staining. In vivo, N-acetylcysteine treatment resulted in improved graft retention at 3 months compared with control (46 versus 17 percent; p = 0.027). N-Acetylcysteine-treated grafts demonstrated less fibrosis and inflammation, and a 33 percent increase in adipocyte density compared with controls (p < 0.001) that was not associated with increased vascularity. CONCLUSION: These findings provide proof of principle for the addition of N-acetylcysteine to tumescent harvest solution in the clinical setting to optimize fat graft yields.
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Acetilcisteína/farmacología , Adipocitos/efectos de los fármacos , Adipocitos/trasplante , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Tejido Adiposo/trasplante , Animales , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Citometría de Flujo , Supervivencia de Injerto , Masculino , Ratones , Ratones Endogámicos C57BL , Distribución Aleatoria , Valores de Referencia , Trasplante de Células Madre , Recolección de Tejidos y Órganos , Trasplante AutólogoRESUMEN
Metastatic lesions are responsible for over 90% of breast cancer associated deaths. Therefore, strategies that target metastasis are of particular interest. This study examined the efficacy of natural killer T (NKT) cell activation as a post-surgical immunotherapy in a mouse model of metastatic breast cancer. Following surgical resection of orthotopic 4T1 mammary carcinoma tumors, BALB/c mice were treated with NKT cell activating glycolipid antigens (α-GalCer, α-C-GalCer or OCH) or α-GalCer-loaded dendritic cells (DCs). Low doses of glycolipids transiently reduced metastasis but did not increase survival. A high dose of α-GalCer enhanced overall survival, but was associated with increased toxicity and mortality at early time points. Treatment with α-GalCer-loaded DCs limited tumor metastasis, prolonged survival, and provided curative outcomes in â¼45% of mice. However, survival was not increased further by additional DC treatments or co-transfer of expanded NKT cells. NKT cell activation via glycolipid-loaded DCs decreased the frequency and immunosuppressive activity of myeloid derived suppressor cells (MDSCs) in tumor-resected mice. In vitro, NKT cells were resistant to the immunosuppressive effects of MDSCs and were able to reverse the inhibitory effects of MDSCs on T cell proliferation. NKT cell activation enhanced antitumor immunity in tumor-resected mice, increasing 4T1-specific cytotoxic responses and IFNγ production from natural killer (NK) cells and CD8+ T cells. Consistent with increased tumor immunity, mice surviving to day 150 were resistant to a second tumor challenge. This work provides a clear rationale for manipulating NKT cells to target metastatic disease.