RESUMO
Tumor progression and response to treatment are highly affected by interactions between cancer cells and the tumor microenvironment (TME). Many of the soluble factors and signaling receptors involved in this crosstalk are shed by a disintegrin and metalloproteinases (ADAMs). Upregulation of ADAM15 has been linked to worse survival in cancer patients and a tumor-promoting function both in vitro and in murine cancer models. Although ADAM15 has been involved in cell-cell and cell-extracellular matrix interactions, its role in the crosstalk between cancer cells and the TME in vivo remains unexplored. Therefore, we aimed to understand how ADAM15 regulates the cell composition of the TME and how it affects tumor progression. Here, we showed an upregulation of ADAM15 in tumor tissues from rectal cancer patients. Subcutaneous injection of wildtype and ADAM15-knockout CT26 colon cancer cells in syngeneic mice confirmed the protumorigenic role of ADAM15. Profiling of tumors revealed higher immune cell infiltration and cancer cell apoptosis in the ADAM15-deficient tumors. Specifically, loss of ADAM15 led to a reduced number of granulocytes and higher infiltration of antigen-presenting cells, including dendritic cells and macrophages, as well as more T cells. Using in vitro assays, we confirmed the regulatory effect of ADAM15 on macrophage migration and identified ADAM15-derived CYR61 as a potential molecular mediator of this effect. Based on these findings, we speculate that targeting ADAM15 could increase the infiltration of immune cells in colorectal tumors, which is a prerequisite for effective immunotherapy.
Assuntos
Neoplasias Colorretais , Microambiente Tumoral , Humanos , Camundongos , Animais , Transdução de Sinais , Movimento Celular , Neoplasias Colorretais/genética , Proteínas de Membrana , Proteínas ADAM/genéticaRESUMO
AIMS: Glioblastomas are heterogeneous tumours with a rich tumour microenvironment particularly comprised of tumour-associated microglia/macrophages (TAMs), but also containing a population of dedifferentiated/stem-like glioblastoma cells. Both cell populations contribute to tumour aggressiveness and immune evasion through the actions of various signalling molecules. The scavenger and pattern recognition receptor CD204 is associated with a pro-tumourigenic phenotype of TAMs and has a negative prognostic value. Our objective was to investigate the possible interaction between TAMs and dedifferentiated glioblastoma cells and characterise the myeloid phenotype of CD204-enriched glioblastomas. METHODS: Double immunohistochemistry and cell counting was performed on eight glioblastoma samples to estimate the expression and interaction level between dedifferentiated/stem-like tumour cells and TAMs. Using the NanoString technology, myeloid transcriptome profiling was performed on 46 glioblastomas, which had been selected based on their protein expression levels of CD204 and ionised calcium-binding adaptor molecule-1 (IBA1). The results were validated by immunohistochemistry and in silico gene expression analyses. RESULTS: TAMs especially CD204+ TAMs accumulated in perivascular and perinecrotic niches in close proximity to podoplanin+ glioblastoma cells. Gene profiling revealed that CD204-enriched glioblastoma has a unique signature with upregulation of genes related to hypoxia, angiogenesis and invasion, including interleukin-6. The gene signature favoured a poor prognosis in patients with glioblastoma. CONCLUSIONS: This is the first study to characterise the role of CD204 in the myeloid microenvironment of glioblastoma. Our results support the unfavourable prognostic impact of CD204 and suggest that CD204 and interleukin-6 could serve as targets for re-education of TAMs and potentiation of current anti-glioma therapy.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Inflamação/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Microglia/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/patologia , Feminino , Glioblastoma/patologia , Humanos , Imuno-Histoquímica , Inflamação/patologia , Macrófagos/patologia , Masculino , Microglia/patologia , Pessoa de Meia-Idade , Prognóstico , Microambiente TumoralRESUMO
BACKGROUND: Sodium fluorescein (fluorescein) crosses a disrupted blood-brain barrier similarly to gadolinium contrast in contrast-enhancing cerebral tumors. When exposed to light with 560 nm wavelength during surgery, fluorescein emits a yellow-green fluorescent light that can be visualized through an operating microscope equipped with an appropriate emission filter. The distribution of the fluorescence correlates with the contrast on a gadolinium contrast-enhanced MRI. OBJECTIVE: The objective of this single-center retrospective study was to investigate if the use of fluorescein would increase the extent of resection and to examine if fluorescein guided resection influences postoperative neurological status. METHODS: During the study period from August 2014 to August 2018, 117 patients were operated for cerebral metastases. Of these, 56 operations were guided by fluorescein and 61 by traditional white light. All patients had an early postoperative MRI within 72 h after surgery. RESULTS: The use of fluorescein increased the extent of resection in patients with cerebral metastases. The use of fluorescein was not associated with increased postoperative sequelae or neurological damage regardless of underlying primary cancer. CONCLUSION: Fluorescein is a helpful supplement in the neurosurgical treatment of cerebral metastases.
Assuntos
Neoplasias Encefálicas , Neoplasias Supratentoriais , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/cirurgia , Fluoresceína , Corantes Fluorescentes , Humanos , Procedimentos Neurocirúrgicos , Estudos Retrospectivos , Neoplasias Supratentoriais/cirurgiaRESUMO
Parkinson's disease is a neurodegenerative disease resulting in degeneration of midbrain dopaminergic neurons. Exploratory studies using human foetal tissue or predifferentiated stem cells have suggested that intracerebral transplantation of dopaminergic precursor cells may become an effective treatment for patients with Parkinson's disease. However, strategies for dopaminergic stem cell differentiation vary widely in efficiency, and better methods still need to be developed. Hypoxia Inducible Factor 1 (HIF-1) is a transcription factor involved in the regulation of genes important for cellular adaption to hypoxia and low glucose supply. HIF-1 is to a large degree regulated by the availability of oxygen as in its presence, the subunit HIF-1α is degraded by HIF prolyl hydroxylase enzymes (HPHs). Stabilization of HIF-1α through inhibition of HPHs has been shown to increase dopaminergic differentiation of stem cells and to protect dopaminergic neurons against neurotoxins. This study investigated the effects of noncompetitive (FG-0041) and competitive (Compound A and JNJ-42041935) HIF-1α stabilizing compounds on the dopaminergic differentiation of human neural stem cells. Treatment with all HPH inhibitors at high oxygen tension (20%) resulted in HIF-1α stabilization as assessed by immunocytochemistry for HIF-1α and detection of increased levels of vascular endothelial growth factor in the conditioned culture medium. Following 10 days of HIF-1α stabilization, the cultures displayed a slightly reduced proliferative activity and significantly increased relative levels of tyrosine hydroxylase-positive dopaminergic neurons. In conclusion, HIF-1α stabilization may represent a promising strategy for the generation of dopaminergic neurons intended for use in experimental in vitro studies and cell replacement therapies.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Inibidores de Prolil-Hidrolase/farmacologia , Benzimidazóis/farmacologia , Linhagem Celular , Feto , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Mesencéfalo/citologia , Doença de Parkinson , Fenantrolinas/farmacologia , Pirazóis/farmacologiaRESUMO
BACKGROUND: We have previously identified tissue inhibitor of metalloproteinases-1 (TIMP-1) as a prognostic marker in glioblastomas. TIMP-1 has been associated with chemotherapy resistance, and CD63, a known TIMP-1-binding protein, has been suggested to be responsible for this effect. The aim of this study was to assess CD63 expression in astrocytomas focusing on the prognostic potential of CD63 alone and in combination with TIMP-1. METHODS: CD63 expression was investigated immunohistochemically in a cohort of 111 astrocytomas and correlated to tumor grade and overall survival by semi-quantitative scoring. CD63 expression in tumor-associated microglia/macrophages was examined by double-immunofluorescence with ionized calcium-binding adapter molecule 1 (Iba1). The association between CD63 and TIMP-1 was investigated using previously obtained TIMP-1 data from our astrocytoma cohort. Cellular co-expression of TIMP-1 and CD63 as well as TIMP-1 and the tumor stem cell-related markers CD133 and Sox2 was investigated with immunofluorescence. TIMP-1 and CD63 protein interaction was detected by an oligonucleotide-based proximity ligation assay and verified using co-immunoprecipitation. RESULTS: The expression of CD63 was widely distributed in astrocytomas with a significantly increased level in glioblastomas. CD63 levels did not significantly correlate with patient survival at a protein level, and CD63 did not augment the prognostic significance of TIMP-1. Up to 38% of the CD63+ cells expressed Iba1; however, Iba1 did not appear to impact the prognostic value of CD63. A significant correlation was found between TIMP-1 and CD63, and the TIMP-1 and CD63 proteins were co-expressed at the cellular level and located in close molecular proximity, suggesting that TIMP-1 and CD63 could be co-players in glioblastomas. Some TIMP-1+ cells expressed CD133 and Sox2. CONCLUSION: The present study suggests that CD63 is highly expressed in glioblastomas and that TIMP-1 and CD63 interact. CD63 does not add to the prognostic value of TIMP-1. Co-expression of TIMP-1 and stem cell markers as well as the wide expression of CD63 might suggest a role for TIMP-1 and CD63 in glioblastoma stemness.
Assuntos
Biomarcadores Tumorais/metabolismo , Membrana Celular/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Tetraspanina 30/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Seguimentos , Humanos , Imunoprecipitação , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Prognóstico , Taxa de Sobrevida , Adulto JovemRESUMO
Shifting the balance away from tumor-mediated immune suppression toward tumor immune rejection is the conceptual foundation for a variety of immunotherapy efforts currently being tested. These efforts largely focus on activating antitumor immune responses but are confounded by multiple immune cell populations, including myeloid-derived suppressor cells (MDSCs), which serve to suppress immune system function. We have identified immune-suppressive MDSCs in the brains of GBM patients and found that they were in close proximity to self-renewing cancer stem cells (CSCs). MDSCs were selectively depleted using 5-flurouracil (5-FU) in a low-dose administration paradigm, which resulted in prolonged survival in a syngeneic mouse model of glioma. In coculture studies, patient-derived CSCs but not nonstem tumor cells selectively drove MDSC-mediated immune suppression. A cytokine screen revealed that CSCs secreted multiple factors that promoted this activity, including macrophage migration inhibitory factor (MIF), which was produced at high levels by CSCs. Addition of MIF increased production of the immune-suppressive enzyme arginase-1 in MDSCs in a CXCR2-dependent manner, whereas blocking MIF reduced arginase-1 production. Similarly to 5-FU, targeting tumor-derived MIF conferred a survival advantage to tumor-bearing animals and increased the cytotoxic T cell response within the tumor. Importantly, tumor cell proliferation, survival, and self-renewal were not impacted by MIF reduction, demonstrating that MIF is primarily an indirect promoter of GBM progression, working to suppress immune rejection by activating and protecting immune suppressive MDSCs within the GBM tumor microenvironment. Stem Cells 2016;34:2026-2039.
Assuntos
Neoplasias Encefálicas/imunologia , Glioblastoma/imunologia , Evasão da Resposta Imune , Fatores Inibidores da Migração de Macrófagos/metabolismo , Células Supressoras Mieloides/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Arginase/metabolismo , Neoplasias Encefálicas/patologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Feminino , Glioblastoma/patologia , Humanos , Evasão da Resposta Imune/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Nus , Células Supressoras Mieloides/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
The gene expression time-course of repeated challenge of contact allergy (CA) remains largely unknown. Therefore, using diphenylcyclopropenone (DPCP) as model allergen in healthy humans we set out to examine: (i) the monotonous and complex gene expression time-course trajectories following repeated DPCP challenges to find the predominant gene expression pattern, (ii) the time-course of cell infiltration following repeated DPCP challenges and (iii) the transcriptome of a repeated CA exposure model. We obtained punch biopsies from control and DPCP-exposed skin from ten DPCP sensitized individuals at 5-6 monthly elicitation challenges. Biopsies were used for microarray gene expression profiling, histopathology and immunohistochemical staining. Validation of microarray data by qRT-PCR was performed on 15 selected genes. Early gene expression time points were also validated in an independent data set. An increasing and decreasing trend in gene expression followed by a plateau was predominantly observed during repeated DPCP challenges. Immune responses reached a plateau after two challenges histopathologically, immunohistochemically and in the time-course gene expression analysis. Transcriptional responses over time revealed a Th1/Th17 polarization as three upstream regulators (IFN-γ, IL-1 and IL-17) activated most of the top upregulated genes. Of the latter genes, 9 of 10 were the same throughout the time course. Excellent correlations between array and PCR data were observed. The transcriptional responses to DPCP over time followed a monotonous pattern. This response pattern confirms and supports the newly reported clinical time-course observations in de novo-sensitized individuals showing a plateau response, and thus, there is concordance between clinical response, histopathology, immunohistochemistry and microarray gene expression in volunteers de novo-sensitized to DPCP.
Assuntos
Dermatite Alérgica de Contato/genética , Dermatite Alérgica de Contato/imunologia , Expressão Gênica , Pele/metabolismo , Transcriptoma , Adulto , Biópsia , Ciclopropanos , Dermatite Alérgica de Contato/etiologia , Dermatite Alérgica de Contato/patologia , Feminino , Perfilação da Expressão Gênica , Voluntários Saudáveis , Humanos , Interferon gama/metabolismo , Interleucina-1/metabolismo , Interleucina-17/metabolismo , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Pele/patologia , Células Th1/imunologia , Células Th17/imunologia , Fatores de Tempo , Adulto JovemRESUMO
BACKGROUND: Glioblastoma is the most frequent and most malignant brain tumor with the patients having a median survival of only 14.6 months. Although glioblastoma patients are treated with surgery, radiation and chemotherapy recurrence is inevitable. A stem-like population of radio- and chemoresistant brain tumor-initiating cells combined with the invasive properties of the tumors is believed to be critical for treatment resistance. In the present study, the aim was to investigate the effect of a novel therapeutic strategy using the lysosomotropic detergent siramesine on glioblastomas. METHODS: Standard glioma cell lines and patient-derived spheroids cultures with tumor-initiating stem-like cells were used to investigate effects of siramesine on proliferation and cell death. Responsible mechanisms were investigated by inhibitors of caspases and cathepsins. Effects of siramesine on migrating tumor cells were investigated by a flat surface migration assay and by implanting spheroids into organotypic rat brain slice cultures followed by confocal time-lapse imaging. Finally the effect of siramesine was investigated in an orthotopic mouse glioblastoma model. Results obtained in vitro and in vivo were confirmed by immunohistochemical staining of histological sections of spheroids, spheroids in brain slice cultures and tumors in mice brains. RESULTS: The results showed that siramesine killed standard glioma cell lines in vitro, and loss of acridine orange staining suggested a compromised lysosomal membrane. Co-treatment of the cell lines with inhibitors of caspases and cathepsins suggested differential involvement in cell death. Siramesine caused tumor cell death and reduced secondary spheroid formation of patient-derived spheroid cultures. In the flat surface migration model siramesine caused tumor cell death and inhibited tumor cell migration. This could not be reproduced in the organotypic three dimensional spheroid-brain slice culture model or in the mice xenograft model. CONCLUSIONS: In conclusion the in vitro results obtained with tumor cells and spheroids suggest a potential of lysosomal destabilizing drugs in killing glioblastoma cells, but siramesine was without effect in the organotypic spheroid-brain slice culture model and the in vivo xenograft model.
Assuntos
Glioblastoma/tratamento farmacológico , Indóis/administração & dosagem , Lisossomos/efeitos dos fármacos , Recidiva Local de Neoplasia/tratamento farmacológico , Compostos de Espiro/administração & dosagem , Animais , Morte Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Glioblastoma/patologia , Humanos , Lisossomos/patologia , Camundongos , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gliomas are among the most lethal cancers, being highly resistant to both chemo- and radiotherapy. The expression of junctional adhesion molecule-A (JAM-A) was recently identified on the surface of stem cell-like brain tumor-initiating cells and suggested to function as a unique glioblastoma niche adhesion factor influencing the tumorigenic potential of brain tumor-initiating cells. We have recently identified high JAM-A expression to be associated with poor outcome in glioblastomas, and our aim was to further investigate the expression of JAM-A in gliomas focusing especially on the prognostic value in WHO grade II and III gliomas. JAM-A protein expression was evaluated by immunohistochemistry and advanced quantitative image analysis with continuous estimates of staining intensity. The JAM-A antibody stained tumor cell membranes and cytoplasm to various extent in different glioma subtypes, and the intensity was higher in glioblastomas than low-grade gliomas. We could not detect an association with overall survival in patients with grade II and III tumors. Double-immunofluorescence stainings in glioblastomas revealed co-expression of JAM-A with CD133, SOX2, nestin, and GFAP in tumor cells as well as some co-expression with the microglial/macrophage marker IBA-1. In conclusion, JAM-A expression was higher in glioblastomas compared to low-grade gliomas and co-localized with recognized stem cell markers suggesting an association of JAM-A with glioma aggressiveness. No significant association between JAM-A expression and overall survival was found in grade II and III gliomas. Further research is needed to determine the function and clinical impact of JAM-A in gliomas.
Assuntos
Neoplasias Encefálicas/metabolismo , Moléculas de Adesão Celular/metabolismo , Glioma/metabolismo , Receptores de Superfície Celular/metabolismo , Antígeno AC133/metabolismo , Adulto , Biomarcadores Tumorais/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proteínas de Ligação ao Cálcio , Estudos de Coortes , Proteínas de Ligação a DNA/metabolismo , Feminino , Expressão Gênica , Proteína Glial Fibrilar Ácida/metabolismo , Glioma/genética , Glioma/patologia , Humanos , Masculino , Proteínas dos Microfilamentos , Pessoa de Meia-Idade , Gradação de Tumores , Nestina , Prognóstico , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Análise de SobrevidaRESUMO
Glioblastoma multiforme (GBM) is the most frequent malignant primary brain tumor. A major reason for the overall median survival being only 14.6 months is migrating tumor cells left behind after surgery. Another major reason is tumor cells having a so-called cancer stem cell phenotype being therefore resistant towards traditional chemo- and radiotherapy. A group of novel molecular targets are microRNAs (miRNAs). MiRNAs are small non-coding RNAs exerting post-transcriptional regulation of gene expression. The aim of this study was to identify differentially expressed miRNAs in migrating GBM cells using serum-free stem cell conditions. We used patient-derived GBM spheroid cultures for a novel serum-free migration assay. MiRNA expression of migrating tumor cells isolated at maximum migration speed was compared with corresponding spheroids using an OpenArray Real-Time PCR System. The miRNA profiling revealed 30 miRNAs to be differentially expressed. In total 13 miRNAs were upregulated and 17 downregulated in migrating cells compared to corresponding spheroids. The three most deregulated miRNAs, miR-1227 (up-regulated), miR-32 (down-regulated) and miR-222 (down-regulated), were experimentally overexpressed. A non-significantly increased migration rate was observed after miR-1227 overexpression. A significantly reduced migration rate was observed after miR-32 and miR-222 overexpression. In conclusion a shift in microRNA profile upon glioma cell migration was identified using an assay avoiding serum-induced migration. Both the miRNA profiling and the functional validation suggested that miR-1227 may be associated with increased migration and miR-32 and miR-222 with decreased migration. These miRNAs may represent potential novel targets in migrating glioma cells.
Assuntos
Neoplasias Encefálicas/metabolismo , Movimento Celular , Glioblastoma/metabolismo , MicroRNAs/metabolismo , Meios de Cultura Livres de Soro , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Esferoides Celulares , Células Tumorais CultivadasRESUMO
The 2016 update of the WHO classification has introduced an integrated diagnostic approach that incorporates both tumor morphology and molecular information. This conceptual change has far-reaching implications, especially for neuropathologists who are in the forefront of translating molecular markers to routine diagnostic use. Adult diffuse glioma is a prototypic example for a group of tumors that underwent substantial regrouping, and it represents a major workload for surgical neuropathologists. Hence, we conducted a survey among members of the European Confederation of Neuropathological Societies (Euro-CNS) in order to assess 1) the extent to which molecular markers have already been incorporated in glioma diagnoses, 2) which molecular techniques are in daily use, and 3) to set a baseline for future surveys in this field. Based on 130 responses from participants across 40 nations neuropathologists uniformly rate molecular marker testing as highly relevant and already incorporate molecular information in their diagnostic assessments. At the same time however, the survey documents substantial differences in access to crucial biomarkers and molecular techniques across geographic regions and within individual countries. Concerns are raised concerning the validity of test assays with MGMT, 1p19q, and ATRX; being perceived as most problematic. Neuropathologists advocate the need for international harmonization of standards and consensus guidelines, and the majority is willing to actively engage in interlaboratory trials aiming at quality control (Figure 1).â©.
Assuntos
Neoplasias Encefálicas/diagnóstico , Glioma/diagnóstico , Oncologia/normas , Neurologia/normas , Patologia Clínica/normas , Adulto , Biomarcadores Tumorais/normas , Humanos , Inquéritos e QuestionáriosRESUMO
Over-expressed microRNAs (miRs) are promising new targets in glioblastoma (GBM) therapy. Inhibition of over-expressed miRs has been shown to diminish GBM proliferation, invasion and angiogenesis, indicating a significant therapeutic potential. However, the methods utilized for miR inhibition have had low translational potential. In clinical trials convection-enhanced delivery (CED) has been applied for local delivery of compounds in the brain. The aim of this study was to determine if safe and efficient miR inhibition was possible by CED of an anti-miR. We used a highly invasive GBM orthotopic xenograft model and targeted a well-validated miR, let-7a, with a 2'-O-methoxyethyl anti-miR with a combined phosphodiester/phosphorothioate backbone to establish an initial proof of concept. In vitro, anti-let-7a was delivered unassisted to the patient-derived T87 glioblastoma spheroid culture. In vivo, anti-let-7a or saline were administered by CED into orthotopic T87-derived tumors. After 1 month of infusion, tumors were removed and tumor mRNA levels of the target-gene High-mobility group AT-hook 2 (HMGA2) were determined. In vitro, 5 days inhibition was superior to 1 day at de-repressing the let-7a target HMGA2 and the inhibition was stable for 24 h. In vivo, anti-miR integrity was preserved in the pumps and no animals showed signs of severe adverse effects attributable to the anti-miR treatment. HMGA2 tumor level was significantly de-repressed in the anti-miR treated animals. The results showed-as an initial proof of concept-that miRs can be efficiently inhibited using CED delivery of anti-miR. The next step is to apply CED for anti-miR delivery focusing on key oncogenic miRs.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/terapia , Glioblastoma/terapia , MicroRNAs/metabolismo , Animais , Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Convecção , Sistemas de Liberação de Medicamentos , Glioblastoma/metabolismo , Glioma/patologia , Proteína HMGA2/genética , Proteína HMGA2/metabolismo , Humanos , Camundongos , Camundongos Nus , MicroRNAs/genética , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/uso terapêutico , RNA Mensageiro/metabolismo , Estatísticas não Paramétricas , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been associated with poor prognosis and resistance towards chemotherapy in several cancer forms. In a previous study we found an association between a low TIMP-1 tumor immunoreactivity and increased survival for glioblastoma patients, when compared to moderate and high TIMP-1 tumor immunoreactivity. The aim of the present study was to further evaluate TIMP-1 as a biomarker in gliomas by studying TIMP-1 gene copy numbers by fluorescence in situ hybridization (FISH) on 33 glioblastoma biopsies and by measuring levels of TIMP-1 in plasma obtained pre-operatively from 43 patients (31 gliomas including 21 glioblastomas) by enzyme-linked immunosorbent assay (ELISA). The results showed TIMP-1 gene copy numbers per cell ranging from 1 to 5 and the TIMP-1/CEN-X ratio ranging between 0.7 and 1.09, suggesting neither amplification nor loss of the TIMP-1 gene. The TIMP-1 protein levels measured in plasma were not significantly higher than TIMP-1 levels measured in healthy subjects. No correlation was identified between TIMP-1 tumor cell immunoreactivities and the TIMP-1 gene copy numbers or the plasma TIMP-1 levels. In conclusion, high immunohistochemical TIMP-1 protein levels in glioblastomas were not caused by TIMP-1 gene amplification and TIMP-1 in plasma was low and not directly related to tumor TIMP-1 immunoreactivity. The study suggests that TIMP-1 immunohistochemistry is the method of choice for future clinical studies evaluating TIMP-1 as a biomarker in glioblastomas.
Assuntos
Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/genética , Glioblastoma/sangue , Glioblastoma/genética , Inibidor Tecidual de Metaloproteinase-1/sangue , Inibidor Tecidual de Metaloproteinase-1/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Variações do Número de Cópias de DNA , Dinamarca , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Análise Serial de TecidosRESUMO
Glioblastomas always recur despite surgery, radiotherapy and chemotherapy. A key player in the therapeutic resistance may be immature tumor cells with stem-like properties (TSCs) escaping conventional treatment. A group of promising molecular targets are microRNAs (miRs). miRs are small non-coding RNAs exerting post-transcriptional regulation of gene expression. In this study we aimed to identify over-expressed TSC-related miRs potentially amenable for therapeutic targeting. We used non-differentiated glioblastoma spheroid cultures (GSCs) containing TSCs and compared these to xenografts using a NanoString nCounter platform. This revealed 19 over-expressed miRs in the non-differentiated GSCs. Additionally, non-differentiated GSCs were compared to neural stem cells (NSCs) using a microarray platform. This revealed four significantly over-expressed miRs in the non-differentiated GSCs in comparison to the NSCs. The three most over-expressed miRs in the non-differentiated GSCs compared to xenografts were miR-126, -137 and -128. KEGG pathway analysis suggested the main biological function of these over-expressed miRs to be cell-cycle arrest and diminished proliferation. To functionally validate the profiling results suggesting association of these miRs with stem-like properties, experimental over-expression of miR-128 was performed. A consecutive limiting dilution assay confirmed a significantly elevated spheroid formation in the miR-128 over-expressing cells. This may provide potential therapeutic targets for anti-miRs to identify novel treatment options for GBM patients.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Células Cultivadas , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Xenoenxertos , Humanos , Masculino , Análise em Microsséries , Transplante de Neoplasias , Células-Tronco Neurais/metabolismo , Ratos Nus , Esferoides Celulares/transplanteRESUMO
BACKGROUND: Tumors in the central nervous system (CNS) comprise a heterogeneous group of tumors with different treatment strategies and prognoses. Current treatment regimens are based on studies on patients mainly younger than 70 years. The aim of the present study was to analyze and describe trends in incidence, mortality, prevalence, and relative survival in Denmark from 1980 to 2012 focusing on patients older than 70 years. MATERIAL AND METHODS: Tumors in the CNS were defined as ICD-10 codes C70-72, D32-33 and D42-43. Data with comparable data on cancer incidence, mortality, prevalence and relative survival derived from the NORDCAN database were delivered from the Danish Cancer Registry and the Danish Cause of Death Registry with follow-up for death or emigration until the end of 2013. RESULTS: During the period 1980-2012 the number of patients with CNS tumors increased from 603 to 1378 patients. The increase is seen mainly in the elderly patients, and especially in women aged 84-89 and 90 + at the time of diagnosis. During the same time period, the mortality rates increased within all age groups, most significantly in patients aged 70 years or older. This may reflect an increased focus on and identification of these patients. Noteworthy; the number of patients living with a CNS tumor increased from 2952 in 1980 to 12 147 patients in 2010. CONCLUSION: This study suggests that the current treatment strategies in general may have improved survival in patients with CNS tumors, but in order to improve survival further in the increasing group of elderly patients more knowledge about treatment of these patients is needed.
Assuntos
Neoplasias do Sistema Nervoso Central/epidemiologia , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Neoplasias do Sistema Nervoso Central/mortalidade , Neoplasias do Sistema Nervoso Central/patologia , Dinamarca/epidemiologia , Glioma/epidemiologia , Humanos , Incidência , Masculino , Prevalência , Prognóstico , Sistema de Registros , Taxa de SobrevidaRESUMO
AIM: Oral contraceptive use influences the risk for certain cancers. However, few studies have examined any link with risk of central nervous system tumours. We investigated the association between hormonal contraceptive use and glioma risk among premenopausal women in a population-based setting. METHODS: Using national administrative and health registries in Denmark to conduct a nationwide case-control study, we identified all women ages 15 to 49 years with a first time diagnosis of histologically verified glioma between 2000 and 2009. Each case was age-matched to eight population controls using risk set sampling. Based on prescription data, exposure until 2 years prior to the index date was categorized according to hormonal contraceptive type, i.e. combined oestrogen-progestagen or progestagen only, and duration of use (<1, 1 to <5, ≥5 years). We used conditional logistic regression to compute odds ratios (ORs) with 95% confidence intervals (CIs) for glioma associated with hormonal contraceptive use, adjusting for potential confounders. RESULTS: We identified 317 cases and 2126 controls. Ever use of hormonal contraceptive was associated with an OR of 1.5 (95% CI 1.2, 2.0) and the OR increased with duration of use (long term, ≥5 years: OR 1.9; 95% CI 1.2, 2.9). The association between long term hormonal contraceptive use and glioma risk was most pronounced for progestagen only therapy (OR 2.4; 95% CI 1.1, 5.1), especially when this regimen constituted the sole hormonal contraceptive therapy (OR 4.1; 95% CI 0.8, 20.8). CONCLUSION: Long term hormonal contraceptive use may increase the risk of glioma.
Assuntos
Neoplasias Encefálicas/induzido quimicamente , Anticoncepcionais Orais Hormonais/efeitos adversos , Glioma/induzido quimicamente , Adolescente , Adulto , Neoplasias Encefálicas/epidemiologia , Estudos de Casos e Controles , Dinamarca/epidemiologia , Feminino , Glioma/epidemiologia , Humanos , Pessoa de Meia-Idade , Razão de Chances , Risco , Adulto JovemRESUMO
BACKGROUND: Crosstalk between the immune system in the brain and the periphery may contribute to the long-term outcome both in experimental and clinical stroke. Although, the immune defense collectin surfactant protein-D (SP-D) is best known for its role in pulmonary innate immunity, SP-D is also known to be involved in extrapulmonary modulation of inflammation in mice. We investigated whether SP-D affected cerebral ischemic infarction and ischemia-induced inflammatory responses in mice. METHODS: The effect of SP-D was studied by comparing the size of ischemic infarction and the inflammatory and astroglial responses in SP-D knock out (KO) and wild type (WT) mice subjected to permanent middle cerebral artery occlusion. SP-D mRNA production was assessed in isolated cerebral arteries and in the whole brain by PCR, and SP-D protein in normal appearing and ischemic human brain by immunohistochemistry. Changes in plasma SP-D and TNF were assessed by ELISA and proximity ligation assay, respectively. RESULTS: Infarct volumetric analysis showed that ablation of SP-D had no effect on ischemic infarction one and five days after induction of ischemia. Further, ablation of SP-D had no effect on the ischemia-induced increase in TNF mRNA production one day after induction of ischemia; however the TNF response to the ischemic insult was affected at five days. SP-D mRNA was not detected in parenchymal brain cells in either naïve mice or in mice subjected to focal cerebral ischemia. However, SP-D mRNA was detected in middle cerebral artery cells in WT mice and SP-D protein in vascular cells both in normal appearing and ischemic human brain tissue. Measurements of the levels of SP-D and TNF in plasma in mice suggested that levels were unaffected by the ischemic insult. Microglial-leukocyte and astroglial responses were comparable in SP-D KO and WT mice. CONCLUSIONS: SP-D synthesis in middle cerebral artery cells is consistent with SP-D conceivably leaking into the infarcted area and affecting local cytokine production. However, there was no SP-D synthesis in parenchymal brain cells and ablation of SP-D had no effect on ischemic cerebral infarction.
Assuntos
Infarto Cerebral/metabolismo , Proteína D Associada a Surfactante Pulmonar/sangue , Proteína D Associada a Surfactante Pulmonar/deficiência , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Antígeno CD11b/metabolismo , Artérias Cerebrais/metabolismo , Artérias Cerebrais/patologia , Infarto Cerebral/etiologia , Infarto Cerebral/patologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Infarto da Artéria Cerebral Média/complicações , Antígenos Comuns de Leucócito/metabolismo , Leucócitos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , Microglia/metabolismo , Proteína D Associada a Surfactante Pulmonar/genética , Fatores de Tempo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Human leukocyte antigen-G (HLA-G) is a nonclassical major histocompatibility complex (MHC) class I molecule involved in immune tolerance processes, playing an important role in the maintenance of the semi-allogeneic fetus. Although HLA-G expression is restricted in normal tissues, it is broadly expressed in malignant tumors and may favor tumor immune escape. We analyzed HLA-G protein and mRNA expression in tumor samples from patients with glioblastoma collected in France, Denmark, and Brazil. We found HLA-G protein expression in 65 of 108 samples and mRNA in 20 of 21 samples. The absence of HLA-G protein expression was associated with a better long-term survival rate. The mechanisms underlying HLA-G gene expression were investigated in glioma cell lines U251MG, D247MG, and U138MG. Induction of HLA-G transcriptional activity was dependent of 5-aza-2'-deoxycytidine treatment and enhanced by interferon-γ. HLA-G protein expression was observed in U251MG cells only. These cells exhibited a permissive chromatin state at the HLA-G gene promoter and the highest levels of induced HLA-G transcriptional activity following 5-aza-2'-deoxycytidine treatment. Several antigen-presenting machinery components were up-regulated in U251MG cells after demethylating and IFN-γ treatments, suggesting an effect on the up-regulation of HLA-G cell surface expression. Therefore, because of its role in tumor tolerance, HLA-G found to be expressed in glioblastoma samples should be taken into consideration in clinical studies on the pathology and in the design of therapeutic strategies to prevent its expression in HLA-G-negative tumors.
Assuntos
Azacitidina/análogos & derivados , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Glioblastoma/genética , Glioblastoma/imunologia , Antígenos HLA-G/genética , Interferon gama/farmacologia , Acetilação/efeitos dos fármacos , Idoso , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/imunologia , Azacitidina/farmacologia , Biópsia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Decitabina , Feminino , Glioblastoma/patologia , Antígenos HLA-G/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Inclusão em Parafina , Prognóstico , Regiões Promotoras Genéticas/genética , Análise de Sobrevida , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Microglobulina beta-2/metabolismoRESUMO
Glioblastoma is the most common primary brain tumor in adults, characterized by an inherent aggressivity and resistance to treatment leading to poor prognoses. While some resistance mechanisms have been elucidated, a deeper understanding of these mechanisms is needed to increase therapeutic efficacy. In this study we first discovered glial-cell derived neurotrophic factor (GDNF) to be upregulated in patient-derived glioblastoma spheroid cultures after chemotherapeutic temozolomide treatment, through RNA-Seq experiments. Therefore, we investigated the role of the GDNF/GDNF receptor alpha 1 (GFRA1) signaling pathway as a resistance mechanism to chemotherapy with temozolomide and lomustine, as well as irradiation using patient-derived glioblastoma spheroid cultures. With qPCR experiments we showed a consistent upregulation of GDNF and its primary receptor GFRA1 following all three lines of treatment. Moreover, CRISPR/Cas9 knock-outs of GDNF in two patient-derived models sensitized these cells to chemotherapy treatment, but not radiotherapy. The increased sensitivity was completely reversed by the addition of exogeneous GDNF, confirming the key role of this factor in chemoresistance. Finally, a CRISPR KO of GFRA1 demonstrated a similar increased sensitivity to temozolomide and lomustine treatment, as well as radiotherapy. Together, our findings support the role of the GDNF/GFRA1 signaling pathway in glioblastoma chemo and radioresistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Glioblastoma , Tolerância a Radiação , Transdução de Sinais , Temozolomida , Glioblastoma/metabolismo , Glioblastoma/genética , Glioblastoma/radioterapia , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Resistencia a Medicamentos Antineoplásicos/genética , Temozolomida/farmacologia , Tolerância a Radiação/genética , Tolerância a Radiação/efeitos dos fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Lomustina/farmacologia , Esferoides Celulares/metabolismo , Esferoides Celulares/efeitos dos fármacosRESUMO
Glioblastoma (GBM) is an aggressive brain tumor with poor prognosis. Although immunotherapy is being explored as a potential treatment option for patients with GBM, it is unclear whether systemic immunotherapy can reach and modify the tumor microenvironment in the brain. We evaluated immune characteristics in patients receiving the anti-PD-1 immune checkpoint inhibitor nivolumab 1 week prior to surgery, compared with control patients receiving salvage resection without prior nivolumab treatment. We observed saturating levels of nivolumab bound to intratumorally and tissue-resident T cells in the brain, implicating saturating levels of nivolumab reaching brain tumors. Following nivolumab treatment, significant changes in T-cell activation and proliferation were observed in the tumor-resident T-cell population, and peripheral T cells upregulated chemokine receptors related to brain homing. A strong nivolumab-driven upregulation in compensatory checkpoint inhibition molecules, i.e., TIGIT, LAG-3, TIM-3, and CTLA-4, was observed, potentially counteracting the treatment effect. Finally, tumor-reactive tumor-infiltrating lymphocytes (TIL) were found in a subset of nivolumab-treated patients with prolonged survival, and neoantigen-reactive T cells were identified in both TILs and blood. This indicates a systemic response toward GBM in a subset of patients, which was further boosted by nivolumab, with T-cell responses toward tumor-derived neoantigens. Our study demonstrates that nivolumab does reach the GBM tumor lesion and enhances antitumor T-cell responses both intratumorally and systemically. However, various anti-inflammatory mechanisms mitigate the clinical efficacy of the anti-PD-1 treatment.