RESUMEN
Endostatin (ES) can effectively inhibit neovascularization in most solid tumors and has the potential to make oxygen delivery more efficient and increase the efficacy of radiotherapy (RT). With a short half-life, ES is mainly administered systemically, which leads to low intake in tumor tissue and often toxic systemic side effects. In this study, we used hyaluronic acid-tyramine as a carrier to synthesize an ES-loaded hydrogel drug (ES/HA-Tyr) that can be injected locally. ES/HA-Tyr has a longer half-life and fewer systemic toxic side effects, and it exerts a better anti-angiogenic effect and anti-tumor effect with RT. In vitro, ES/HA-Tyr showed sustained release in the release assay and a stronger ability to inhibit the proliferation of human umbilical vascular endothelial cells (HUVECs) in the MTT assay; it exhibited a more potent effect against HUVEC invasion and a stronger anti-angiogenic effect on HUVECs in tube formation. In vivo, ES/HA-Tyr increased local drug concentration, decreased blood drug concentration, and caused less systemic toxicity. Further, ES/HA-Tyr effectively reduced tumor microvessel density, increased tumor pericyte coverage, decreased tumor hypoxia, and increased RT response. ES/HA-Tyr + RT also had increased anti-tumor and anti-angiogenic effects in Lewis lung cancer (LLC) xenograft models. In conclusion, ES/HA-Tyr showed sustained release, lower systemic toxicity, and better anti-tumor effects than ES. In addition, ES/HA-Tyr + RT enhanced anti-angiogenic effects, reduced tumor hypoxia, and increased the efficacy of RT in LLC-bearing mice.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Endostatinas/administración & dosificación , Ácido Hialurónico , Hidrogeles , Neoplasias Pulmonares/tratamiento farmacológico , Tiramina , Inhibidores de la Angiogénesis/farmacología , Animales , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/patología , Quimioradioterapia , Portadores de Fármacos , Liberación de Fármacos , Endostatinas/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Técnicas In Vitro , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/patología , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: To investigate whether the vascular collapse in tumors by conventional dose rate (CONV) irradiation (IR) would also occur by the ultra-high dose rate FLASH IR. METHODS AND MATERIALS: Lewis lung carcinoma (LLC) cells were subcutaneously implanted in mice. This was followed by CONV or FLASH IR at 15 Gy. Tumors were harvested at 6 or 48 hours after IR and stained for CD31, phosphorylated myosin light chain (p-MLC), γH2AX (a surrogate marker for DNA double strand break), intracellular reactive oxygen species (ROS), or immune cells such as myeloid and CD8α T cells. Cell lines were irradiated with CONV IR for Western blot analyses. ML-7 was intraperitoneally administered daily to LLC-bearing mice for 7 days before 15 Gy CONV IR. Tumors were similarly harvested and analyzed. RESULTS: By immunostaining, we observed that CONV IR at 6 hours resulted in constricted vessel morphology, increased expression of p-MLC, and much higher numbers of γH2AX-positive cells in tumors, which were not observed with FLASH IR. Mechanistically, MLC activation by ROS is unlikely, because FLASH IR produced significantly more ROS than CONV IR in tumors. In vitro studies demonstrated that ML-7, an inhibitor of MLC kinase, abrogated IR-induced γH2AX formation and disappearance kinetics. Lastly, we observed that CONV IR when combined with ML-7 produced some effects similar to FLASH IR, including reduction in the vasculature collapse, fewer γH2AX-positive cells, and increased immune cell influx to the tumors. CONCLUSIONS: FLASH IR produced novel changes in the tumor microenvironment that were not observed with CONV IR. We believe that MLC activation in tumors may be responsible for some of the microenvironmental changes differentially regulated between CONV and FLASH IR.
Asunto(s)
Carcinoma Pulmonar de Lewis/radioterapia , Cadenas Ligeras de Miosina/efectos de la radiación , Microambiente Tumoral/efectos de la radiación , Animales , Azepinas/administración & dosificación , Vasos Sanguíneos/patología , Vasos Sanguíneos/efectos de la radiación , Linfocitos T CD8-positivos/citología , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/metabolismo , Histonas/metabolismo , Histonas/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Cadenas Ligeras de Miosina/antagonistas & inhibidores , Cadenas Ligeras de Miosina/metabolismo , Naftalenos/administración & dosificación , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/efectos de la radiación , Radioterapia/métodos , Dosificación Radioterapéutica , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/efectos de la radiaciónRESUMEN
BACKGROUND: Conventional therapeutic approaches for tumor angiogenesis, which are primarily focused on the inhibition of active angiogenesis to starve cancerous cells, target the vascular endothelial growth factor signaling pathway. This aggravates hypoxia within the tumor core and ultimately leads to increased tumor proliferation and metastasis. To overcome this limitation, we developed nanoparticles with antiseptic activity that target tumor vascular abnormalities. METHODS: Ferritin-based protein C nanoparticles (PCNs), known as TFG and TFMG, were generated and tested in Lewis lung carcinoma (LLC) allograft and MMTV-PyMT spontaneous breast cancer models. Immunohistochemical analysis was performed on tumor samples to evaluate the tumor vasculature. Western blot and permeability assays were used to explore the role and mechanism of the antitumor effects of PCNs in vivo. For knocking down proteins of interest, endothelial cells were transfected with siRNAs. Statistical analysis was performed using one-way ANOVA followed by post hoc Dunnett's multiple comparison test. RESULTS: PCNs significantly inhibited hypoxia and increased pericyte coverage, leading to the inhibition of tumor growth and metastasis, while increasing survival in LLC allograft and MMTV-PyMT spontaneous breast cancer models. The coadministration of cisplatin with PCNs induced a synergistic suppression of tumor growth by improving drug delivery as evidenced by increased blood prefusion and decreased vascular permeability. Moreover, PCNs altered the immune cell profiles within the tumor by increasing cytotoxic T cells and M1-like macrophages with antitumor activity. PCNs induced PAR-1/PAR-3 heterodimerization through EPCR occupation and PAR-1 activation, which resulted in Gα13-RhoA-mediated-Tie2 activation and stabilized vascular tight junctions via the Akt-FoxO3a signaling pathway. CONCLUSIONS: Cancer treatment targeting the tumor vasculature by inducing antitumor immune responses and enhancing the delivery of a chemotherapeutic agent with PCNs resulted in tumor regression and may provide an effective therapeutic strategy.
Asunto(s)
Apoferritinas/uso terapéutico , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Nanopartículas/uso terapéutico , Neovascularización Patológica/tratamiento farmacológico , Proteína C/uso terapéutico , Receptor TIE-2/fisiología , Remodelación Vascular/efectos de los fármacos , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Antineoplásicos Alquilantes/administración & dosificación , Antineoplásicos Alquilantes/uso terapéutico , Apoferritinas/administración & dosificación , Bevacizumab/uso terapéutico , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/patología , Hipoxia de la Célula/efectos de los fármacos , Cisplatino/administración & dosificación , Cisplatino/uso terapéutico , Técnicas de Cocultivo , Sistemas de Liberación de Medicamentos , Sinergismo Farmacológico , Células Endoteliales/efectos de los fármacos , Femenino , Masculino , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Neoplasias/fisiología , Pericitos/metabolismo , Proteína C/administración & dosificación , Organismos Libres de Patógenos Específicos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
Tumor blood vessels have leaky and low blood flow properties, which lead to hypoxia and low nutrient levels in the tumor tissue area known as the tumor microenvironment (TME). We reported that the prolyl-hydroxylase (PHD) inhibitor Roxadustat normalized tumor blood vessels, improved tumor tissue perfusion, and re-oxygenated the tumor tissue. Recently, several PHD inhibitors including Roxadustat, Daprodustat, Molidustat, and Vadadustat, were evaluated in clinical trials and approved for treating renal anemia. In this study, we showed that PHD inhibitors reconstituted tumor blood vessels and improved the TME, and some agents exhibited differential effects on tumors in a mouse model.
Asunto(s)
Carcinoma Pulmonar de Lewis/irrigación sanguínea , Glicina/análogos & derivados , Isoquinolinas/farmacología , Ácidos Picolínicos/farmacología , Inhibidores de Prolil-Hidroxilasa/farmacología , Animales , Carcinoma Pulmonar de Lewis/metabolismo , Glicina/farmacología , Masculino , Ratones Endogámicos C57BL , Consumo de Oxígeno/efectos de los fármacos , Células Tumorales Cultivadas , Microambiente Tumoral/efectos de los fármacosRESUMEN
Blocking the immune evasion mechanism of tumor cells has become an attractive means for treating cancers. However, the usage of a drug such as nivolumab (αPD-1), which blocks programmed cell death protein 1 (PD-1), turned out to be only effective against certain types of cancer. Especially, vascular abnormal structures of which deter delivery route by leakage and cause the poor perfusion were considered to be environment unfavorable to T cells and immune checkpoint blockade (ICB) delivery within the tumor microenvironment (TME). Herein, we report stabilization of tumor blood vessels by endothelial dysfunctional blocker CU06-1004, which modified the TME and showed synergistic effects with immunotherapy anti-PD-1 antibody. CU06-1004 combination therapy consistently prolonged the survival of tumor-bearing mice by decreasing tumor growth. T-cell infiltration increased in the tumors of the combination group, with cytotoxic CD8+ T cell activity within the tumor parenchyma upregulated compared with anti-PD-1 monotherapy. Tumor inhibition was associated with reduced hypoxia and reduced vessel density in the central region of the tumor. These effects correlated significantly with enhanced expression of IFN gamma and PD-L1 in tumors. Taken together, our findings suggest that CU06-1004 is a potential candidate drug capable of improving therapeutic efficacy of anti-PD-1 through beneficial changes in the TME.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Endotelio Vascular/efectos de los fármacos , Inhibidores de Puntos de Control Inmunológico/farmacocinética , Inmunoterapia/métodos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Saponinas/farmacología , Linfocitos T Citotóxicos/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Animales , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/farmacocinética , Antineoplásicos Inmunológicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Apoptosis/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Endotelio Vascular/inmunología , Inhibidores de Puntos de Control Inmunológico/administración & dosificación , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/fisiopatología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Saponinas/administración & dosificación , Saponinas/uso terapéutico , Linfocitos T Citotóxicos/inmunología , Carga Tumoral/efectos de los fármacos , Escape del Tumor/efectos de los fármacosRESUMEN
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, which function as transcription factors. Among them, PPARß/δ is highly expressed in endothelial cells. Pharmacological activation with PPARß/δ agonists had been shown to increase their angiogenic properties. PPARß/δ has been suggested to be involved in the regulation of the angiogenic switch in tumor progression. However, until now, it is not clear to what extent the expression of PPARß/δ in tumor endothelium influences tumor progression and metastasis formation. We addressed this question using transgenic mice with an inducible conditional vascular-specific overexpression of PPARß/δ. Following specific over-expression of PPARß/δ in endothelial cells, we induced syngenic tumors. We observed an enhanced tumor growth, a higher vessel density, and enhanced metastasis formation in the tumors of animals with vessel-specific overexpression of PPARß/δ. In order to identify molecular downstream targets of PPARß/δ in the tumor endothelium, we sorted endothelial cells from the tumors and performed RNA sequencing. We identified platelet-derived growth factor receptor beta (Pdgfrb), platelet-derived growth factor subunit B (Pdgfb), and the tyrosinkinase KIT (c-Kit) as new PPARß/δ -dependent molecules. We show here that PPARß/δ activation, regardless of its action on different cancer cell types, leads to a higher tumor vascularization which favors tumor growth and metastasis formation.
Asunto(s)
Carcinoma Pulmonar de Lewis/irrigación sanguínea , Células Endoteliales/metabolismo , Neovascularización Patológica/metabolismo , PPAR delta/metabolismo , PPAR-beta/metabolismo , Animales , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Línea Celular Tumoral , Progresión de la Enfermedad , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , PPAR delta/agonistas , PPAR-beta/agonistas , Proteínas Proto-Oncogénicas c-kit/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Tiazoles/farmacologíaRESUMEN
Systemic inhibition of Dll4 has been shown to thoroughly reduce cancer metastasis. The exact cause of this effect and whether it is endothelial mediated remains to be clarified. Therefore, we proposed to analyze the impact of endothelial Dll4 loss-of-function on metastasis induction on three early steps of the metastatic process, regulation of epithelial-to-mesenchymal transition (EMT), cancer stem cell (CSC) frequency and circulating tumor cell (CTC) number. For this, Lewis Lung Carcinoma (LLC) cells were used to model mouse tumor metastasis in vivo, by subcutaneous transplantation into endothelial-specific Dll4 loss-of-function mice. We observed that endothelial-specific Dll4 loss-of-function is responsible for the tumor vascular regression that leads to the reduction of tumor burden. It induces an increase in tumoral blood vessel density, but the neovessels are poorly perfused, with increased leakage and reduced perivascular maturation. Unexpectedly, although hypoxia was increased in the tumor, the number and burden of macro-metastasis was significantly reduced. This is likely to be a consequence of the observed reduction in both EMT and CSC numbers caused by the endothelial-specific Dll4 loss-of-function. This multifactorial context may explain the concomitantly observed reduction of the circulating tumor cell count. Furthermore, our results suggest that endothelial Dll4/Notch-function mediates tumor hypoxia-driven increase of EMT. Therefore, it appears that endothelial Dll4 may constitute a promising target to prevent metastasis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas de Unión al Calcio/fisiología , Transición Epitelial-Mesenquimal , Metástasis de la Neoplasia/prevención & control , Células Neoplásicas Circulantes , Células Madre Neoplásicas/fisiología , Animales , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/patología , Hipoxia de la Célula , Células Endoteliales/fisiología , Ratones , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Receptores Notch/fisiología , Transducción de Señal , Carga TumoralRESUMEN
The tumor microenvironment regulates cancer initiation, progression and response to treatment. In particular, the immature tumor vasculature may impede drugs from reaching tumor cells at a lethal concentration. We and others have shown that radiation therapy (RT) induces pericyte recruitment, resembling vascular normalization. Here, we asked whether radiation-induced vascular remodeling translates into improved tissue distribution and efficacy of chemotherapy. First, RT induced vascular remodeling, accompanied by decreased hypoxia and/or increased Hoechst perfusion in prostate PC3 and LNCaP and Lewis lung carcinoma. These results were independent of the RT regimen, respectively 10â¯×â¯2â¯Gy and 2â¯×â¯12â¯Gy, suggesting a common effect. Next, using doxorubicin as a fluorescent reporter, we observed that RT improves intra-tumoral chemotherapy distribution. These effects were not hindered by anti-angiogenic sunitinib. Moreover, sub-optimal doses of doxorubicin had almost no effect alone, but significantly delayed tumor growth after RT. These data demonstrate that RT favors the efficacy of chemotherapy by improving tissue distribution, and could be an alternative chemosensitizing strategy.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/terapia , Quimioradioterapia , Doxorrubicina/farmacología , Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/terapia , Dosis de Radiación , Remodelación Vascular/efectos de la radiación , Animales , Antibióticos Antineoplásicos/metabolismo , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Doxorrubicina/metabolismo , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Desnudos , Neovascularización Patológica , Células PC-3 , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Distribución Tisular , Carga Tumoral/efectos de los fármacos , Carga Tumoral/efectos de la radiación , Hipoxia Tumoral , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The lack of response to treatment in most lung cancer patients suggests the value of broadening the benefit of anti-PD-1/PD-L1 monotherapy. Judicious dosing of antiangiogenic agents such as apatinib (VEGFR2-TKI) can modulate the tumor immunosuppressive microenvironment, which contributes to resistance to anti-PD-1/PD-L1 treatment. We therefore hypothesized that inhibiting angiogenesis could enhance the therapeutic efficacy of PD-1/PD-L1 blockade. Here, using a syngeneic lung cancer mouse model, we demonstrated that low-dose apatinib alleviated hypoxia, increased infiltration of CD8+ T cells, reduced recruitment of tumor-associated macrophages in tumor and decreased TGFß amounts in both tumor and serum. Combining low-dose apatinib with anti-PD-L1 significantly retarded tumor growth, reduced the number of metastases, and prolonged survival in mouse models. Anticancer activity was evident after coadministration of low-dose apatinib and anti-PD-1 in a small cohort of patients with pretreated advanced non-small cell lung cancer. Overall, our work shows the rationale for the treatment of lung cancer with a combination of PD-1/PD-L1 blockade and low-dose apatinib.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/uso terapéutico , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Antígeno B7-H1/metabolismo , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Pulmonar de Lewis/metabolismo , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/metabolismo , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Microambiente Tumoral/efectos de los fármacosRESUMEN
Increased levels of the chemokine CCL2 in cancer patients are associated with poor prognosis. Experimental evidence suggests that CCL2 correlates with inflammatory monocyte recruitment and induction of vascular activation, but the functionality remains open. Here, we show that endothelial Ccr2 facilitates pulmonary metastasis using an endothelial-specific Ccr2-deficient mouse model (Ccr2ecKO). Similar levels of circulating monocytes and equal leukocyte recruitment to metastatic lesions of Ccr2ecKO and Ccr2fl/fl littermates were observed. The absence of endothelial Ccr2 strongly reduced pulmonary metastasis, while the primary tumor growth was unaffected. Despite a comparable cytokine milieu in Ccr2ecKO and Ccr2fl/fl littermates the absence of vascular permeability induction was observed only in Ccr2ecKO mice. CCL2 stimulation of pulmonary endothelial cells resulted in increased phosphorylation of MLC2, endothelial cell retraction, and vascular leakiness that was blocked by an addition of a CCR2 inhibitor. These data demonstrate that endothelial CCR2 expression is required for tumor cell extravasation and pulmonary metastasis. IMPLICATIONS: The findings provide mechanistic insight into how CCL2-CCR2 signaling in endothelial cells promotes their activation through myosin light chain phosphorylation, resulting in endothelial retraction and enhanced tumor cell migration and metastasis.
Asunto(s)
Carcinoma Pulmonar de Lewis/metabolismo , Quimiocina CCL2/metabolismo , Células Endoteliales/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores CCR2/metabolismo , Animales , Permeabilidad Capilar , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/patología , Carcinoma Pulmonar de Lewis/secundario , Movimiento Celular/fisiología , Células Endoteliales/patología , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cadenas Ligeras de Miosina/metabolismo , Metástasis de la NeoplasiaRESUMEN
The transient receptor potential vanilloid 4 (TRPV4) channel is a mechanosensor in endothelial cells (EC) that regulates cyclic strain-induced reorientation and flow-mediated nitric oxide production. We have recently demonstrated that TRPV4 expression is reduced in tumor EC and tumors grown in TRPV4KO mice exhibited enhanced growth and immature leaky vessels. However, the mechanism by which TRPV4 regulates tumor vascular integrity and metastasis is not known. Here, we demonstrate that VE-cadherin expression at the cell-cell contacts is significantly reduced in TRPV4-deficient tumor EC and TRPV4KO EC. In vivo angiogenesis assays with Matrigel of varying stiffness (700-900â¯Pa) revealed a significant stiffness-dependent reduction in VE-cadherin-positive vessels in Matrigel plugs from TRPV4KO mice compared with WT mice, despite an increase in vessel growth. Further, syngeneic Lewis Lung Carcinomatumor experiments demonstrated a significant decrease in VE-cadherin positive vessels in TRPV4KO tumors compared with WT. Functionally, enhanced tumor cell metastasis to the lung was observed in TRPV4KO mice. Our findings demonstrate that TRPV4 channels regulate tumor vessel integrity by maintaining VE-cadherin expression at cell-cell contacts and identifies TRPV4 as a novel target for metastasis.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Movimiento Celular , Células Endoteliales/metabolismo , Uniones Intercelulares/metabolismo , Neoplasias Pulmonares/irrigación sanguínea , Mecanotransducción Celular , Neovascularización Patológica , Canales Catiónicos TRPV/metabolismo , Animales , Antígenos CD/genética , Cadherinas/genética , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/secundario , Células Endoteliales/patología , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Uniones Intercelulares/genética , Uniones Intercelulares/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Invasividad Neoplásica , Canales Catiónicos TRPV/deficiencia , Canales Catiónicos TRPV/genéticaRESUMEN
Angiogenesis is coordinated by VEGF and Notch signaling. DLL4-induced Notch signaling inhibits tip cell formation and vessel branching. To ensure proper Notch signaling, receptors and ligands are clustered at adherens junctions. However, little is known about factors that control Notch activity by influencing the cellular localization of Notch ligands. Here, we show that the multiple PDZ domain protein (MPDZ) enhances Notch signaling activity. MPDZ physically interacts with the intracellular carboxyterminus of DLL1 and DLL4 and enables their interaction with the adherens junction protein Nectin-2. Inactivation of the MPDZ gene leads to impaired Notch signaling activity and increased blood vessel sprouting in cellular models and the embryonic mouse hindbrain. Tumor angiogenesis was enhanced upon endothelial-specific inactivation of MPDZ leading to an excessively branched and poorly functional vessel network resulting in tumor hypoxia. As such, we identified MPDZ as a novel modulator of Notch signaling by controlling ligand recruitment to adherens junctions.
Asunto(s)
Carcinoma Pulmonar de Lewis/irrigación sanguínea , Proteínas Portadoras/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Melanoma Experimental/irrigación sanguínea , Proteínas de la Membrana/metabolismo , Neovascularización Patológica/patología , Neovascularización Fisiológica , Receptores Notch/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Unión al Calcio , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Receptores Notch/genética , Transducción de SeñalRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: The medicinal mushroom Antrodia cinnamomea has been used to treat cancer but its anti-angiogenic effects have not been studied in detail. AIM OF THE STUDY: The main objective of this study was to determine the molecular mechanism of activity underlying the anti-angiogenic effects of A. cinnamomea. MATERIALS AND METHODS: The effects of an A. cinnamomea ethanol extract (ACEE) on cell migration and microvessel formation were investigated in endothelial cells in vitro and Matrigel plugs implanted into mice in vivo. Activation of intracellular signaling pathways was examined using Western blotting. Protein expression was assessed using immunohistochemistry in a mouse model of lung metastasis. RESULTS: We show that treatment with ACEE inhibits cell migration and tube formation in human umbilical vein endothelial cells (HUVECs). ACEE suppresses phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and expression of pro-angiogenic kinases in vascular endothelial growth factor (VEGF)-treated HUVECs, in addition to reducing expression of Janus kinase 2 (JAK2) and phosphorylation of signal transducer and activator of transcription 3 (STAT3). ACEE treatment inhibits VEGF-induced microvessel formation in Matrigel plugs in vivo. In addition, ACEE significantly reduces VEGFR2 expression in Lewis lung carcinoma cells and downregulates the expression of cluster of differentiation 31 (CD31) and VEGFR2 in murine lung metastases. CONCLUSION: These results indicate that A. cinnamomea produces anti-angiogenic effects by inhibiting the VEGFR2 signaling pathway.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antrodia/química , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Inhibidores de la Angiogénesis/aislamiento & purificación , Animales , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Movimiento Celular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: Intermedin plays an important role in vascular remodeling and significantly improves blood perfusion, but the precise mechanism remains unclear. Herein, we aimed to define whether vascular lumen enlargement is responsible for the intermedin-increased blood perfusion and explore the underlying cellular and molecular mechanisms. APPROACH AND RESULTS: To study the role of intermedin, we generated the IMD-KO (Adm2-/-) mice using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) system. Intermedin significantly promoted vascular lumen enlargement in vitro (fibrin beads assay) and in vivo (murine retinas), which contributed to the improved blood perfusion in both physiological (retinal) and pathological (tumor) angiogenic models. We designed experiments to calculate the endothelial cell (EC) size and found that the lumen enlargement is because of EC proliferation but not because of a change in cell shape. ECs that construct vessel walls are considered quiescent cells because they are in a state of contact inhibition and show reduced responsiveness to VEGF (vascular endothelial growth factor). Using immunoprecipitation, Western blot assay, and fluorescent microscopy, we found that intermedin induced the formation of a signaling complex containing CRLR (calcitonin receptor-like receptor)/ß-arr1 (ß-arrestin1)/Src in ECs and promoted it internalizing into cytoplasm in a clathrin-dependent manner to activate downstream ERK1/2 (extracellular signal-regulated kinase 1/2). Importantly, this effect was not abrogated by cell-cell contacts of ECs. Through this mechanism, intermedin could reactivate the quiescent ECs to proliferate, resulting in continuous lumen expanding and a more effective blood perfusion. CONCLUSIONS: Our findings suggest a novel mechanism that may explain how quiescent ECs overcome the contact inhibition and regain the ability to proliferate for continuous vascular lumen enlargement.
Asunto(s)
Carcinoma Pulmonar de Lewis/irrigación sanguínea , Proliferación Celular , Senescencia Celular , Neoplasias del Colon/irrigación sanguínea , Células Endoteliales/metabolismo , Neovascularización Patológica , Neovascularización Fisiológica , Neuropéptidos/metabolismo , Vasos Retinianos/metabolismo , Animales , Línea Celular Tumoral , Técnicas de Cocultivo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neuropéptidos/deficiencia , Neuropéptidos/genética , Hormonas Peptídicas/genética , Hormonas Peptídicas/metabolismo , Flujo Sanguíneo Regional , Transducción de Señal , Remodelación VascularRESUMEN
Interactions between different tumors within the same organism have major clinical implications, especially in the context of surgery and metastatic disease. Three main explanatory theories (competition, angiogenesis inhibition, and proliferation inhibition) have been proposed, but precise determinants of the phenomenon remain poorly understood. Here, we formalized these theories into mathematical models and performed biological experiments to test them with empirical data. In syngeneic mice bearing two simultaneously implanted tumors, growth of only one of the tumors was significantly suppressed (61% size reduction at day 15, P < 0.05). The competition model had to be rejected, whereas the angiogenesis inhibition and proliferation inhibition models were able to describe the data. Additional models including a theory based on distant cytotoxic log-kill effects were unable to fit the data. The proliferation inhibition model was identifiable and minimal (four parameters), and its descriptive power was validated against the data, including consistency in predictions of single tumor growth when no secondary tumor was present. This theory may also shed new light on single cancer growth insofar as it offers a biologically translatable picture of how local and global action may combine to control local tumor growth and, in particular, the role of tumor-tumor inhibition. This model offers a depiction of concomitant resistance that provides an improved theoretical basis for tumor growth control and may also find utility in therapeutic planning to avoid postsurgery metastatic acceleration. Cancer Res; 77(18); 5183-93. ©2017 AACR.
Asunto(s)
Carcinoma Pulmonar de Lewis/patología , Proliferación Celular , Modelos Biológicos , Modelos Teóricos , Neovascularización Patológica/patología , Animales , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Masculino , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Células Tumorales CultivadasRESUMEN
OBJECTIVE: Recent genome-wide association studies newly identified the human KIAA1462 gene as a new locus for coronary artery disease. However, the function of the gene product, named JCAD (junctional protein associated with coronary artery disease), is unknown. Because JCAD is expressed at cell-cell junctions in endothelial cells, we hypothesized and tested whether JCAD regulates angiogenic processes in vitro and in vivo. APPROACH AND RESULTS: Cell culture experiments revealed impaired angiogenic ability (proliferation, migration, and cord formation) by the knockdown of JCAD with siRNA (P<0.05 versus control siRNA). We have generated mice lacking JCAD (mKIAA1462-/-) by gene-targeted deletion of JCAD to address in vivo angiogenic function. mKIAA1462-/- mice did not show morphological differences in development of retinal vasculature. Ex vivo aortic ring model demonstrated impaired neovascularization in aorta from mKIAA1462-/- mice than control wild-type mice (P<0.05). Tumor growth was assessed by monitoring tumor volume after the subcutaneous injection of melanoma, LLC (Lewis lung carcinoma), and E0771 cells into the mice. mKIAA1462-/- mice exhibited significantly smaller tumor volume compared with wild-type mice (P<0.001). Histological assessment of the tumor exhibited less smooth muscle actin-positive neovascularization determined by CD31-positive vascular structure in tumor of mKIAA1462-/- mice than wild-type mice, indicating that knockdown of JCAD inhibited the vascular maturation in pathological angiogenic process. CONCLUSIONS: These in vitro and in vivo studies suggest that JCAD has a redundant functional role in physiological angiogenesis but serves a pivotal role in pathological angiogenic process after birth.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Células Endoteliales/metabolismo , Uniones Intercelulares/metabolismo , Neovascularización Patológica , Neovascularización Fisiológica , Neovascularización Retiniana , Animales , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Moléculas de Adhesión Celular/deficiencia , Moléculas de Adhesión Celular/genética , Movimiento Celular , Proliferación Celular , Células Cultivadas , Genotipo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Interferencia de ARN , Transducción de Señal , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Transfección , Carga TumoralRESUMEN
Angiogenesis is important for normal development as well as for tumour growth. However, the molecular and cellular mechanisms underlying angiogenesis are not fully understood, partly because of the lack of a good animal model for imaging. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a bioluminescent reporter protein, Nano-lantern, under the control of Fetal liver kinase 1 (Flk1). Flk1-Nano-lantern BAC Tg mice recapitulated endogenous Flk1 expression in endothelial cells and lymphatic endothelial cells during development and tumour growth. Importantly, bioluminescence imaging of endothelial cells from the aortic rings of Flk1-Nano-lantern BAC Tg mice enabled us to observe endothelial sprouting for 18 hr without any detectable phototoxicity. Furthermore, Flk1-Nano-lantern BAC Tg mice achieved time-lapse luminescence imaging of tumour angiogenesis in freely moving mice with implanted tumours. Thus, this transgenic mouse line contributes a unique model to study angiogenesis within both physiological and pathological contexts.
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Carcinoma Pulmonar de Lewis/diagnóstico por imagen , Células Endoteliales/fisiología , Luciferasas/metabolismo , Proteínas Luminiscentes/metabolismo , Neovascularización Patológica/diagnóstico por imagen , Neovascularización Fisiológica , Proteínas Recombinantes de Fusión/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/metabolismo , Línea Celular Tumoral , Células Endoteliales/metabolismo , Fluorescencia , Luciferasas/genética , Mediciones Luminiscentes/métodos , Proteínas Luminiscentes/genética , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Transgénicos , Microscopía Confocal , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Proteínas Recombinantes de Fusión/genética , Imagen de Lapso de Tiempo/métodos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
BACKGROUND: The inhibition of Delta-like 4 (Dll4)/Notch signaling has been shown to result in excessive, nonfunctional vessel proliferation and significant tumor growth suppression. However, safety concerns emerged with the identification of side effects resulting from chronic Dll4/Notch blockade. Alternatively, we explored the endothelial Dll4 overexpression using different mouse tumor models. METHODS: We used a transgenic mouse model of endothelial-specific Dll4 overexpression, previously produced. Growth kinetics and vascular histopathology of several types of solid tumors was evaluated, namely Lewis Lung Carcinoma xenografts, chemically-induced skin papillomas and RIP1-Tag2 insulinomas. RESULTS: We found that increased Dll4/Notch signaling reduces tumor growth by reducing vascular endothelial growth factor (VEGF)-induced endothelial proliferation, tumor vessel density and overall tumor blood supply. In addition, Dll4 overexpression consistently improved tumor vascular maturation and functionality, as indicated by increased vessel calibers, enhanced mural cell recruitment and increased network perfusion. Importantly, the tumor vessel normalization is not more effective than restricted vessel proliferation, but was found to prevent metastasis formation and allow for increased delivery to the tumor of concomitant chemotherapy, improving its efficacy. CONCLUSIONS: By reducing endothelial sensitivity to VEGF, these results imply that Dll4/Notch stimulation in tumor microenvironment could be beneficial to solid cancer patient treatment by reducing primary tumor size, improving tumor drug delivery and reducing metastization. Endothelial specific Dll4 overexpression thus appears as a promising anti-angiogenic modality that might improve cancer control.
Asunto(s)
Endotelio Vascular/metabolismo , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Neoplasias Experimentales/genética , Neovascularización Patológica/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Unión al Calcio , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Insulinoma/irrigación sanguínea , Insulinoma/genética , Insulinoma/patología , Masculino , Ratones Transgénicos , Metástasis de la Neoplasia , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/patología , Carga Tumoral/genéticaRESUMEN
RATIONALE: Vascular endothelial growth factor (VEGF) is the main driver of angiogenesis and vascular permeability via VEGF receptor 2 (VEGFR2), whereas lymphangiogenesis signals are transduced by VEGFC/D via VEGFR3. VEGFR3 also regulates sprouting angiogenesis and blood vessel growth, but to what extent VEGFR3 signaling controls blood vessel permeability remains unknown. OBJECTIVE: To investigate the role of VEGFR3 in the regulation of VEGF-induced vascular permeability. METHODS AND RESULTS: Long-term global Vegfr3 gene deletion in adult mice resulted in increased fibrinogen deposition in lungs and kidneys, indicating enhanced vascular leakage at the steady state. Short-term deletion of Vegfr3 in blood vascular endothelial cells increased baseline leakage in various tissues, as well as in tumors, and exacerbated vascular permeability in response to VEGF, administered via intradermal adenoviral delivery or through systemic injection of recombinant protein. VEGFR3 gene silencing upregulated VEGFR2 protein levels and phosphorylation in cultured endothelial cells. Consistent with elevated VEGFR2 activity, vascular endothelial cadherin showed reduced localization at endothelial cell-cell junctions in postnatal retinas after Vegfr3 deletion, or after VEGFR3 silencing in cultured endothelial cells. Furthermore, concurrent deletion of Vegfr2 prevented VEGF-induced excessive vascular leakage in mice lacking Vegfr3. CONCLUSIONS: VEGFR3 limits VEGFR2 expression and VEGF/VEGFR2 pathway activity in quiescent and angiogenic blood vascular endothelial cells, thereby preventing excessive vascular permeability.
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Permeabilidad Capilar , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Células Endoteliales/metabolismo , Pulmón/irrigación sanguínea , Vasos Retinianos/metabolismo , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Uniones Adherentes/metabolismo , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Carcinoma Pulmonar de Lewis/metabolismo , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Femenino , Genotipo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica , Neovascularización Fisiológica , Fenotipo , Vasos Retinianos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Uniones Estrechas/metabolismo , Transfección , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/deficiencia , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/deficiencia , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Tumor angiogenesis is the key process in tumor growth and metastasis, and transfers essential nutrients for solid tumor. Inhibition of tumor angiogenesis has been recognized as a more effective anti-cancer strategy for NSCLC and has acquired certain therapeutic effects. IDO has non-immune functions including regulating tumor angiogenesis and IDO dysregulation in cancer pathogenesis has been valued. Erianin is a natural product isolated from Dendrobium chrysotoxum Lindl. The antitumor activity of erianin in many kinds of cancers had been demonstrated in previous studies. In this study, we demonstrated that IDO could promote the attachment of 2LL cells, the ability of migration, invasion and VM formation, as well as the tubules forming ability of HUVECs. We also find that erianin suppressed expression and enzyme ability of IDO and erianin could inhibit IDO-induced metastasis and invasion ability of 2LL cells significantly. Erianin not only blocked IDO-induced tube formation of HUVECs, but also suppressed VM formation of 2LL-IDO cells. What's more, we examined that Erianin might play its role in angiogenesis through down-regulating phosphorylation of JAK2/STAT3, inhibiting its downstream target genes MMP-2/-9 and some inflammatory mediators (COX-2, HIF-1α and IL-6), which were all induced by IDO. All these results indicated that erianin had anti-angiogenesis ability and could inhibit the expresison of IDO to prevent and treat the malignant tumors.