RESUMEN
Bevacizumab, a VEGF-targeting monoclonal antibody, may trigger an infiltrative growth pattern in glioblastoma. We investigated this pattern using both a human specimen and rat models. In the human specimen, a substantial fraction of infiltrating tumor cells were located along perivascular spaces in close relationship with endothelial cells. Brain xenografts of U87MG cells treated with bevacizumab were smaller than controls (p = 0.0055; Student t-test), however, bands of tumor cells spread through the brain farther than controls (p < 0.001; Student t-test). Infiltrating tumor Cells exhibited tropism for vascular structures and propensity to form tubules and niches with endothelial cells. Molecularly, bevacizumab triggered an epithelial to mesenchymal transition with over-expression of the receptor Plexin Domain Containing 1 (PLXDC1). These results were validated using brain xenografts of patient-derived glioma stem-like cells. Enforced expression of PLXDC1 in U87MG cells promoted brain infiltration along perivascular spaces. Importantly, PLXDC1 inhibition prevented perivascular infiltration and significantly increased the survival of bevacizumab-treated rats. Our study indicates that bevacizumab-induced brain infiltration is driven by vascular endothelium and depends on PLXDC1 activation of tumor cells.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Bevacizumab/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Endotelio/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Proteínas de Neoplasias/metabolismo , Receptores de Superficie Celular/metabolismo , Adulto , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Bevacizumab/uso terapéutico , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/patología , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Técnicas de Cocultivo , Resistencia a Antineoplásicos , Células Endoteliales , Endotelio/citología , Endotelio/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Masculino , Proteínas de Neoplasias/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Desnudas , Receptores de Superficie Celular/genética , Análisis de Supervivencia , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We hypothesized that in glioblastoma recurring after radiotherapy, a condition whereby the brain endothelium undergoes radiation-induced senescence, tumor cells with endothelial phenotype may be relevant for tumor neovascularization. Matched glioblastoma samples obtained at primary surgery and at surgery for tumor recurrence after radiotherapy, all expressing epidermal growth factor receptor variant III (EGFRvIII), were assessed by a technique that combines fluorescent in situ hybridization (FISH) for the EGFR/CEP7 chromosomal probe with immunostaining for endothelial cells (CD31) and activated pericytes (α Smooth Muscle Actin). Five EGFRvIII-expressing paired primary/recurrent glioblastoma samples, in which the tumor cells showed EGFR/CEP7 amplification, were then assessed by CD31 and α Smooth Muscle Actin immunofluorescence. In glomeruloid bodies, the ratio between CD31+ cells with amplified EGFR/CEP7 signal and the total CD31+ cells was 0.23 ± 0.09 (mean ± sem) and 0.63 ± 0.07 in primary tumors and in recurrent ones, respectively (p < 0.002, Student-t test). In capillaries, the ratio of CD31+ cells with amplified EGFR/CEP7 over the total CD31+ cells lining the capillary lumen was 0.21 ± 0.06 (mean ± sem) and 0.42 ± 0.07 at primary surgery and at recurrence, respectively (p < 0.005, Student-t test). Expression of α Smooth Muscle Actin by cells with EGFR/CEP7 amplification was not observed. Then, in glioblastoma recurring after radiotherapy, where the brain endothelium suffers from radiation-induced cell senescence, tumor-derived endothelium plays a role in neo-vascularization.
Asunto(s)
Neoplasias Encefálicas/patología , Transdiferenciación Celular/fisiología , Células Endoteliales/patología , Glioblastoma/patología , Recurrencia Local de Neoplasia/patología , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Células Endoteliales/metabolismo , Células Endoteliales/efectos de la radiación , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glioblastoma/genética , Glioblastoma/radioterapia , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Células Endoteliales de la Vena Umbilical Humana/efectos de la radiación , Humanos , Hibridación Fluorescente in Situ , Ratones , Recurrencia Local de Neoplasia/genéticaRESUMEN
Functionally relevant markers of glioblastoma stem-like cells (GSCs) have potential for therapeutic targeting to treat this aggressive disease. Here we used generation and screening of thousands of monoclonal antibodies to search for receptors and signaling pathways preferentially enriched in GSCs. We identified integrin α7 (ITGA7) as a major laminin receptor in GSCs and in primary high-grade glioma specimens. Analyses of mRNA profiles in comprehensive datasets revealed that high ITGA7 expression negatively correlated with survival of patients with both low- and high-grade glioma. In vitro and in vivo analyses showed that ITGA7 plays a key functional role in growth and invasiveness of GSCs. We also found that targeting of ITGA7 by RNAi or blocking mAbs impaired laminin-induced signaling, and it led to a significant delay in tumor engraftment plus a strong reduction in tumor size and invasion. Our data, therefore, highlight ITGA7 as a glioblastoma biomarker and candidate therapeutic target.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/farmacología , Anticuerpos Antineoplásicos/farmacología , Biomarcadores de Tumor/antagonistas & inhibidores , Glioblastoma/tratamiento farmacológico , Cadenas alfa de Integrinas/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Animales , Antígenos CD/metabolismo , Biomarcadores de Tumor/metabolismo , Sistemas de Liberación de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Glioblastoma/metabolismo , Glioblastoma/patología , Células HeLa , Humanos , Cadenas alfa de Integrinas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Mesenchymal stem/stromal cells (MSCs) represent an attractive tool for cell-based cancer therapy mainly because of their ability to migrate to tumors and to release bioactive molecules. However, the impact of MSCs on tumor growth has not been fully established. We previously demonstrated that murine MSCs show a strong tropism towards glioblastoma (GBM) brain xenografts and that these cells are able to uptake and release the chemotherapeutic drug paclitaxel (PTX), maintaining their tropism towards the tumor. Here, we address the therapy-relevant issue of using MSCs from human donors (hMSCs) for local or systemic administration in orthotopic GBM models, including xenografts of patient-derived glioma stem cells (GSCs). METHODS: U87MG or GSC1 cells expressing the green fluorescent protein (GFP) were grafted onto the striatum of immunosuppressed rats. Adipose hMSCs (Ad-hMSCs), fluorescently labeled with the mCherry protein, were inoculated adjacent to or into the tumor. In rats bearing U87MG xenografts, systemic injections of Ad-hMSCs or bone marrow (BM)-hMSCs were done via the femoral vein or carotid artery. In each experiment, either PTX-loaded or unloaded hMSCs were used. To characterize the effects of hMSCs on tumor growth, we analyzed survival, tumor volume, tumor cell proliferation, and microvascular density. RESULTS: Overall, the AD-hMSCs showed remarkable tropism towards the tumor. Intracerebral injection of Ad-hMSCs significantly improved the survival of rats with U87MG xenografts. This effect was associated with a reduction in tumor growth, tumor cell proliferation, and microvascular density. In GSC1 xenografts, intratumoral injection of Ad-hMSCs depleted the tumor cell population and induced migration of resident microglial cells. Overall, PTX loading did not significantly enhance the antitumor potential of hMSCs. Systemically injected Ad- and BM-hMSCs homed to tumor xenografts. The efficiency of hMSC homing ranged between 0.02 and 0.5% of the injected cells, depending both on the route of cell injection and on the source from which the hMSCs were derived. Importantly, systemically injected PTX-loaded hMSCs that homed to the xenograft induced cytotoxic damage to the surrounding tumor cells. CONCLUSIONS: hMSCs have a therapeutic potential in GBM brain xenografts which is also expressed against the GSC population. In this context, PTX loading of hMSCs seems to play a minor role.
Asunto(s)
Proliferación Celular , Glioblastoma/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Animales , Línea Celular Tumoral , Terapia Combinada , Glioblastoma/patología , Humanos , Ratones , Paclitaxel/administración & dosificación , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glioblastoma multiforme (GBM) is the most common and fatal malignant adult primary brain tumor. Currently, the overall prognosis for GBM patients remains poor despite advances in neurosurgery and adjuvant treatments. MicroRNAs (miRNAs) contribute to the pathogenesis of various types of tumor, including GBM. In this study we analyzed the expression of a panel of miRNAs, which are known to be differentially expressed by the brain and GBM tumor, in a collection of patient-derived GBM stem-like cells (GSCs). Notably, the average expression level of miR-135b, was the most downregulated compared to its normal counterpart, suggesting a potential role as anti-oncogene.Restoration of miR-135b in GSCs significantly decreased proliferation, migration and clonogenic abilities. More importantly, miR-135b restoration was able to significantly reduce brain infiltration in mouse models of GBM obtained by intracerebral injection of GSC lines. We identified ADAM12 and confirmed SMAD5 and GSK3ß as miR-135b targets and potential mediators of its effects. The whole transcriptome analysis ascertained that the expression of miR-135b downmodulated additional genes driving key pathways in GBM survival and infiltration capabilities.Our results identify a critical role of miR-135b in the regulation of GBM development, suggesting that miR-135b might act as a tumor-suppressor factor and thus providing a potential candidate for the treatment of GBM patients.
Asunto(s)
Movimiento Celular , Proliferación Celular , Autorrenovación de las Células , Glioblastoma/metabolismo , MicroARNs/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM12 , Animales , Apoptosis , Línea Celular Tumoral , Regulación hacia Abajo , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Genotipo , Glioblastoma/genética , Glioblastoma/patología , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Xenoinjertos , Humanos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos NOD , Ratones SCID , MicroARNs/genética , Invasividad Neoplásica , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/trasplante , Fenotipo , Transducción de Señal , Proteína Smad5/genética , Proteína Smad5/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
INTRODUCTION: The goal of cancer chemotherapy is targeting tumor cells and/or tumor-associated microvessels with the lowest systemic toxicity. Mesenchymal stromal cells (MSCs) are promising vehicles for selective drug delivery due to their peculiar ability to home to pathological tissues. We previously showed that MSCs are able to uptake and subsequently to release the chemotherapeutic compound Paclitaxel (PTX) and to impair the growth of subcutaneous glioblastoma multiforme (GBM) xenografts. Here we used an orthotopic GBM model 1) to assess whether PTX-loaded MSCs (PTX-MSCs) retain a tropism towards the tumor cells in the brain context, and 2) to characterize the cytotoxic damage induced by MSCs-driven PTX release in the tumor microenvironment. METHODS: U87MG GBM cells were fluorescently labeled with the mCherry protein and grafted onto the brain of immunosuppressed rats. In adjacent brain regions, we injected green fluorescent protein-expressing murine MSCs, either loaded with PTX or unloaded. After 1 week survival, the xenografted brain was assessed by confocal microscopy for PTX-induced cell damage. RESULTS: Overall, MSCs showed remarkable tropism towards the tumor. In rats grafted with PTX-MSCs, the nuclei of U87MG cells showed changes that are typically induced by PTX, including multi-spindle mitoses, centrosome number alterations, and nuclear fragmentation. Multi-spindle mitoses resulted in multinucleated cells that were significantly higher in tumors co-grafted with PTX-MSCs than in controls. Nuclear changes did not occur in astrocytes and neurons surrounding the tumor. CONCLUSIONS: MSCs appear particularly suited for anti-neoplastic drug delivery in the brain since PTX-specific damage of GBM cells can be achieved avoiding side effects to the normal tissue.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Trasplante de Células Madre Mesenquimatosas , Paclitaxel/administración & dosificación , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Antineoplásicos Fitogénicos/toxicidad , Línea Celular Tumoral , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Paclitaxel/uso terapéutico , Paclitaxel/toxicidad , Ratas , Ratas WistarRESUMEN
Bone morphogenetic proteins (BMPs), members of the TGF-ß superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v) in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC) Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis.
Asunto(s)
Proteína Morfogenética Ósea 7/uso terapéutico , Células Endoteliales/metabolismo , Glioblastoma/irrigación sanguínea , Neovascularización Patológica/tratamiento farmacológico , Tejido Adiposo/citología , Animales , Proteína Morfogenética Ósea 7/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colágeno/farmacología , Combinación de Medicamentos , Células Endoteliales/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Laminina/farmacología , Masculino , Ratones Desnudos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Neovascularización Patológica/patología , Neovascularización Fisiológica/efectos de los fármacos , Proteoglicanos/farmacología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Experimental evidence indicates that shear stress (SS) exerts a morphogenetic function during cardiac development of mouse and zebrafish embryos. However, the molecular basis for this effect is still elusive. Our previous work described that in adult endothelial cells, SS regulates gene expression by inducing epigenetic modification of histones and activation of transcription complexes bearing acetyltransferase activity. In this study, we evaluated whether SS treatment could epigenetically modify histones and influence cell differentiation in mouse embryonic stem (ES) cells. Cells were exposed to a laminar SS of 10 dyne per cm2/s(-1), or kept in static conditions in the presence or absence of the histone deacetylase inhibitor trichostatin A (TSA). These experiments revealed that SS enhanced lysine acetylation of histone H3 at position 14 (K14), as well as serine phosphorylation at position 10 (S10) and lysine methylation at position 79 (K79), and cooperated with TSA, inducing acetylation of histone H4 and phosphoacetylation of S10 and K14 of histone H3. In addition, ES cells exposed to SS strongly activated transcription from the vascular endothelial growth factor (VEGF) receptor 2 promoter. This effect was paralleled by an early induction of cardiovascular markers, including smooth muscle actin, smooth muscle protein 22-alpha, platelet-endothelial cell adhesion molecule-1, VEGF receptor 2, myocyte enhancer factor-2C (MEF2C), and alpha-sarcomeric actin. In this condition, transcription factors MEF2C and Sma/MAD homolog protein 4 could be isolated from SS-treated ES cells complexed with the cAMP response element-binding protein acetyltransferase. These results provide molecular basis for the SS-dependent cardiovascular commitment of mouse ES cells and suggest that laminar flow may be successfully applied for the in vitro production of cardiovascular precursors.