RESUMO
Cancer immunotherapies have shown substantial clinical activity for a subset of patients with epithelial cancers. Still, technological platforms to study cancer T-cell interactions for individual patients and understand determinants of responsiveness are presently lacking. Here, we establish and validate a platform to induce and analyze tumor-specific T cell responses to epithelial cancers in a personalized manner. We demonstrate that co-cultures of autologous tumor organoids and peripheral blood lymphocytes can be used to enrich tumor-reactive T cells from peripheral blood of patients with mismatch repair-deficient colorectal cancer and non-small-cell lung cancer. Furthermore, we demonstrate that these T cells can be used to assess the efficiency of killing of matched tumor organoids. This platform provides an unbiased strategy for the isolation of tumor-reactive T cells and provides a means by which to assess the sensitivity of tumor cells to T cell-mediated attack at the level of the individual patient.
Assuntos
Leucócitos Mononucleares/citologia , Linfócitos T/imunologia , Idoso , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Técnicas de Cultura de Células , Técnicas de Cocultura , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Humanos , Técnicas In Vitro , Interferon gama/farmacologia , Leucócitos Mononucleares/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
The progressive loss of CNS myelin in patients with multiple sclerosis (MS) has been proposed to result from the combined effects of damage to oligodendrocytes and failure of remyelination. A common feature of demyelinated lesions is the presence of oligodendrocyte precursors (OLPs) blocked at a premyelinating stage. However, the mechanistic basis for inhibition of myelin repair is incompletely understood. To identify novel regulators of OLP differentiation, potentially dysregulated during repair, we performed a genome-wide screen of 1040 transcription factor-encoding genes expressed in remyelinating rodent lesions. We report that approximately 50 transcription factor-encoding genes show dynamic expression during repair and that expression of the Wnt pathway mediator Tcf4 (aka Tcf7l2) within OLPs is specific to lesioned-but not normal-adult white matter. We report that beta-catenin signaling is active during oligodendrocyte development and remyelination in vivo. Moreover, we observed similar regulation of Tcf4 in the developing human CNS and lesions of MS. Data mining revealed elevated levels of Wnt pathway mRNA transcripts and proteins within MS lesions, indicating activation of the pathway in this pathological context. We show that dysregulation of Wnt-beta-catenin signaling in OLPs results in profound delay of both developmental myelination and remyelination, based on (1) conditional activation of beta-catenin in the oligodendrocyte lineage in vivo and (2) findings from APC(Min) mice, which lack one functional copy of the endogenous Wnt pathway inhibitor APC. Together, our findings indicate that dysregulated Wnt-beta-catenin signaling inhibits myelination/remyelination in the mammalian CNS. Evidence of Wnt pathway activity in human MS lesions suggests that its dysregulation might contribute to inefficient myelin repair in human neurological disorders.
Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/fisiopatologia , Regulação da Expressão Gênica no Desenvolvimento , Esclerose Múltipla/fisiopatologia , Bainha de Mielina/metabolismo , Proteínas Wnt/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Humanos , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Fatores de Transcrição TCF/metabolismo , Fator de Transcrição 4 , Fatores de Transcrição/metabolismo , Proteínas Wnt/fisiologia , beta Catenina/metabolismoRESUMO
BACKGROUND: Alzheimer's disease (AD) is the most common form of dementia. AD has a multifactorial disease etiology and is currently untreatable. Multiple genes and molecular mechanisms have been implicated in AD, including ß-amyloid deposition in the brain, neurofibrillary tangle accumulation of hyper-phosphorylated Tau, synaptic failure, oxidative stress and inflammation. Relatively little is known about the role of the blood-brain barriers, especially the blood-cerebrospinal fluid barrier (BCSFB), in AD. The BCSFB is involved in cerebrospinal fluid (CSF) production, maintenance of brain homeostasis and neurodegenerative disorders. RESULTS: Using an Agilent platform with common reference design, we performed a large scale gene expression analysis and functional annotation of the Choroid Plexus Epithelium (CPE), which forms the BCSFB. We obtained 2 groups of freshly frozen Choroid Plexus (CP) of 7 human donor brains each, with and without AD: Braak stages (0-1) and (5-6). We cut CP cryo-sections and isolated RNA from cresyl-violet stained, laser dissected CPE cells. Gene expression results were analysed with T-tests (R) and the knowledge-database Ingenuity. We found statistically significantly altered gene expression data sets, biological functions, canonical pathways, molecular networks and functionalities in AD-affected CPE. We observed specific cellular changes due to increased oxidative stress, such as the unfolded protein response, E1F2 and NRF2 signalling and the protein ubiquitin pathway. Most likely, the AD-affected BCSFB barrier becomes more permeable due to downregulation of CLDN5. Finally, our data also predicted down regulation of the glutathione mediated detoxification pathway and the urea cycle in the AD CPE, which suggest that the CPE sink action may be impaired. Remarkably, the expression of a number of genes known to be involved in AD, such as APP, PSEN1, PSEN2, TTR and CLU is moderate to high and remains stable in both healthy and affected CPE. Literature labelling of our new functional molecular networks confirmed multiple previous (molecular) observations in the AD literature and revealed many new ones. CONCLUSIONS: We conclude that CPE failure in AD exists. Combining our data with those of the literature, we propose the following chronological and overlapping chain of events: increased Aß burden on CPE; increased oxidative stress in CPE; despite continuous high expression of TTR: decreased capability of CPE to process amyloid; (pro-) inflammatory and growth factor signalling by CPE; intracellular ubiquitin involvement, remodelling of CPE tight junctions and, finally, cellular atrophy. Our data corroborates the hypothesis that increased BCSFB permeability, especially loss of selective CLDN5-mediated paracellular transport, altered CSF production and CPE sink action, as well as loss of CPE mediated macrophage recruitment contribute to the pathogenesis of AD.
Assuntos
Doença de Alzheimer/genética , Plexo Corióideo/metabolismo , Perfilação da Expressão Gênica , Anotação de Sequência Molecular , Idoso , Doença de Alzheimer/patologia , Estudos de Casos e Controles , Epitélio/metabolismo , Humanos , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: Repair of myelin injury in multiple sclerosis may fail, resulting in chronic demyelination, axonal loss, and disease progression. As cellular pathways regulated by phosphatase and tensin homologue deleted on chromosome 10 (PTEN; eg, phosphatidylinositol-3-kinase [PI-3K]) have been reported to enhance axon regeneration and oligodendrocyte maturation, we investigated potentially beneficial effects of Pten loss of function in the oligodendrocyte lineage on remyelination. METHODS: We characterized oligodendrocyte numbers and myelin sheath thickness in mice with conditional inactivation of Pten in oligodendrocytes, Olig2-cre, Pten(fl/fl) mice. Using a model of central nervous system demyelination, lysolecithin injection into the spinal cord white matter, we performed short- and long-term lesioning experiments and quantified oligodendrocyte maturation and myelin sheath thickness in remyelinating lesions. RESULTS: During development, we observed dramatic hypermyelination in the corpus callosum and spinal cord. Following white matter injury, however, there was no detectable improvement in remyelination. Moreover, we observed progressive myelin sheath abnormalities and massive axon degeneration in the fasciculus gracilis of mutant animals, as indicated by ultrastructure and expression of SMI-32, amyloid precursor protein, and caspase 6. INTERPRETATION: These studies indicate adverse effects of chronic Pten inactivation (and by extension, activation PI-3K signaling) on myelinating oligodendrocytes and their axonal targets. We conclude that PTEN function in oligodendrocytes is required to regulate myelin thickness and preserve axon integrity. In contrast, PTEN is dispensable during myelin repair, and its inactivation confers no detectable benefit.
Assuntos
Axônios/enzimologia , Bainha de Mielina/metabolismo , Oligodendroglia/enzimologia , PTEN Fosfo-Hidrolase/fisiologia , Fatores Etários , Animais , Axônios/patologia , Encéfalo/patologia , Encéfalo/ultraestrutura , Linhagem Celular Transformada , Doenças Desmielinizantes/enzimologia , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/fisiopatologia , Modelos Animais de Doenças , Deleção de Genes , Humanos , Lisofosfatidilcolinas , Camundongos , Camundongos Transgênicos , Bainha de Mielina/patologia , Bainha de Mielina/ultraestrutura , Fibras Nervosas Mielinizadas/patologia , Fibras Nervosas Mielinizadas/fisiologia , Fibras Nervosas Mielinizadas/ultraestrutura , Oligodendroglia/patologia , Oligodendroglia/fisiologia , Oligodendroglia/ultraestrutura , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Medula Espinal/patologia , Medula Espinal/ultraestruturaRESUMO
Gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC) is a poorly understood disease with limited treatment options. A better understanding of this disease would greatly benefit from the availability of representative preclinical models. Here, we present the potential of tumor organoids, three-dimensional cultures of tumor cells, to model GEP-NEC. We established three GEP-NEC organoid lines, originating from the stomach and colon, and characterized them using DNA sequencing and immunohistochemistry. Organoids largely resembled the original tumor in expression of synaptophysin, chromogranin and Ki-67. Models derived from tumors containing both neuroendocrine and non-neuroendocrine components were at risk of overgrowth by non-neuroendocrine tumor cells. Organoids were derived from patients treated with cisplatin and everolimus and for the three patients studied, organoid chemosensitivity paralleled clinical response. We demonstrate the feasibility of establishing NEC organoid lines and their potential applications. Organoid culture has the potential to greatly extend the repertoire of preclinical models for GEP-NEC, supporting drug development for this difficult-to-treat tumor type.
Assuntos
Neoplasias Intestinais/patologia , Modelos Biológicos , Tumores Neuroendócrinos/patologia , Organoides/patologia , Neoplasias Pancreáticas/patologia , Neoplasias Gástricas/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Everolimo/farmacologia , Everolimo/uso terapêutico , Dosagem de Genes , Humanos , Neoplasias Intestinais/tratamento farmacológico , Neoplasias Intestinais/genética , Antígeno Ki-67/metabolismo , Mutação/genética , Tumores Neuroendócrinos/tratamento farmacológico , Tumores Neuroendócrinos/genética , Organoides/efeitos dos fármacos , Organoides/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Sequenciamento do ExomaRESUMO
Clinical implementation of tumor organoids for personalized medicine requires that pure tumor organoids can be reliably established. Here, we present our experience with organoid cultures from >70 non-small cell lung cancer (NSCLC) samples. We systematically evaluate several methods to identify tumor purity of organoids established from intrapulmonary tumors. Eighty percent of organoids from intrapulmonary lesions have a normal copy number profile, suggesting overgrowth by normal airway organoids (AOs). This is further supported by the failure to detect mutations found in the original tumor in organoids. Histomorphology alone is insufficient to determine tumor purity, but when combined with p63 immunostaining, tumor and normal AOs can be distinguished. Taking into account overgrowth by normal AOs, the establishment rate of pure NSCLC organoids is 17%. Therefore, current methods are insufficient to establish pure NSCLC organoids from intrapulmonary lesions. We discourage their use unless steps are taken to prevent overgrowth by normal AOs.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Organoides/patologia , Medicina de Precisão , Humanos , Neoplasias Pulmonares/genética , Mutação/genética , Medicina de Precisão/métodosRESUMO
T cells are key players in cancer immunotherapy, but strategies to expand tumor-reactive cells and study their interactions with tumor cells at the level of an individual patient are limited. Here we describe the generation and functional assessment of tumor-reactive T cells based on cocultures of tumor organoids and autologous peripheral blood lymphocytes. The procedure consists of an initial coculture of 2 weeks, in which tumor-reactive T cells are first expanded in the presence of (IFNγ-stimulated) autologous tumor cells. Subsequently, T cells are evaluated for their capacity to carry out effector functions (IFNγ secretion and degranulation) after recognition of tumor cells, and their capacity to kill tumor organoids. This strategy is unique in its use of peripheral blood as a source of tumor-reactive T cells in an antigen-agnostic manner. In 2 weeks, tumor-reactive CD8+ T-cell populations can be obtained from ~33-50% of samples from patients with non-small-cell lung cancer (NSCLC) and microsatellite-instable colorectal cancer (CRC). This enables the establishment of ex vivo test systems for T-cell-based immunotherapy at the level of the individual patient.
Assuntos
Técnicas de Cocultura/métodos , Neoplasias/patologia , Organoides/patologia , Linfócitos T/citologia , HumanosRESUMO
There is a clear and unmet clinical need for biomarkers to predict responsiveness to chemotherapy for cancer. We developed an in vitro test based on patient-derived tumor organoids (PDOs) from metastatic lesions to identify nonresponders to standard-of-care chemotherapy in colorectal cancer (CRC). In a prospective clinical study, we show the feasibility of generating and testing PDOs for evaluation of sensitivity to chemotherapy. Our PDO test predicted response of the biopsied lesion in more than 80% of patients treated with irinotecan-based therapies without misclassifying patients who would have benefited from treatment. This correlation was specific to irinotecan-based chemotherapy, however, and the PDOs failed to predict outcome for treatment with 5-fluorouracil plus oxaliplatin. Our data suggest that PDOs could be used to prevent cancer patients from undergoing ineffective irinotecan-based chemotherapy.
Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Organoides/citologia , Antineoplásicos/uso terapêutico , Capecitabina/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Feminino , Fluoruracila/uso terapêutico , Humanos , Irinotecano/uso terapêutico , Oxaliplatina/uso terapêutico , Estudos Prospectivos , Resultado do TratamentoRESUMO
PURPOSE: Chemotherapy-resistance remains a major obstacle to effective anti-cancer treatment. We previously showed that platinum analogs cause the release of two fatty acids. These platinum-induced fatty acids (PIFAs) induced complete chemoresistance in mice, whereas co-administration of a COX-1 inhibitor, indomethacin, prevented PIFA release and significantly enhanced chemosensitivity. To assess the safety of combining indomethacin with platinum-based chemotherapy, and to explore its efficacy and associated PIFA levels, a multi-center phase I trial was conducted. METHODS: The study was comprised of two arms: oxaliplatin plus capecitabine (CAPOX, arm I) and cisplatin plus gemcitabine, capecitabine or 5FU (arm II) in patients for whom these regimens were indicated as standard care. Indomethacin was escalated from 25 to 75 mg TID, using a standard 3 × 3 design per arm, and was administered orally 8 days around chemo-infusion from cycle two onwards. PIFA levels were measured before and after treatment initiation, with and without indomethacin. RESULTS: Thirteen patients were enrolled, of which ten were evaluable for safety analyses. In arm I, no dose-limiting toxicities were observed, and all indomethacin dose levels were well-tolerated. Partial responses were observed in three patients (30%). Indomethacin lowered plasma levels of 12-S-hydroxy-5,8,10-heptadecatrienoic acid (12-S-HHT), whereas 4,7,10,13-hexadecatetraenoic acid (16:4(n-3)) levels were not affected. Only one patient was included in arm II; renal toxicity led to closure of this cohort. CONCLUSIONS: Combined indomethacin and CAPOX treatment is safe and reduces the concentrations of 12-S-HHT, which may be associated with improved chemosensitivity. The recommended phase II dose is 75 mg indomethacin TID given 8 days surrounding standard dosed CAPOX.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Indometacina/farmacologia , Neoplasias/tratamento farmacológico , Compostos Organoplatínicos/farmacologia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ciclo-Oxigenase 1/metabolismo , Inibidores de Ciclo-Oxigenase/uso terapêutico , Relação Dose-Resposta a Droga , Esquema de Medicação , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ácidos Graxos/metabolismo , Feminino , Humanos , Indometacina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Neoplasias/patologia , Compostos Organoplatínicos/uso terapêutico , Estudos Prospectivos , Resultado do TratamentoRESUMO
In the vertebrate brain, much remains to be understood concerning the origin of glial cell diversity and the potential lineage relationships between the various types of glia. Besides astrocytes and myelin-forming oligodendrocytes, other macroglial cell populations are found in discrete areas of the central nervous system (CNS). They share functional features with astrocytes and oligodendrocytes but also display specific characteristics. Such specialized cells, called pituicytes, are located in the neurohypophysis (NH). Our work focuses on the lineage of the pituicytes during rodent development. First, we show that cells identified with a combination of oligodendrocyte precursor cell (OPC) markers are present in the developing rat NH. In culture, neonatal NH progenitors also share major functional characteristics with OPCs, being both migratory and bipotential, i.e. able to give rise to type 2 astrocytes and oligodendrocytes. We then observe that, either in vitro or after transplantation into myelin-deficient Shiverer brain, pieces of NH generate myelinating oligodendrocytes, confirming the oligodendrogenic potentiality of NH cells. However, no mature oligodendrocyte can be found in the NH. This led us to hypothesize that the OPCs present in the developing NH might be generating other glial cells, especially the pituicytes. Consistent with this hypothesis, the OPCs appear during NH development before pituicytes differentiate. Finally, we establish a lineage relationship between olig1+ cells, most likely OPCs, and the pituicytes by fate-mapping experiments using genetically engineered mice. This constitutes the first demonstration that OPCs generate glial cells other than oligodendrocytes in vivo.
Assuntos
Oligodendroglia/metabolismo , Neuro-Hipófise/crescimento & desenvolvimento , Hipófise/citologia , Células-Tronco/metabolismo , Animais , Animais Recém-Nascidos , Movimento Celular , Células Cultivadas , Corantes , Citometria de Fluxo , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Mutantes Neurológicos , Neuroglia/fisiologia , Neuro-Hipófise/citologia , Ratos , Ratos WistarRESUMO
Within the motoneuron precursor (pMN) domain of the developing spinal cord, the bHLH transcription factor, Olig2, plays critical roles in pattern formation and the generation of motor neuron and oligodendrocyte precursors. How are the multiple functions of Olig2 regulated? We have isolated a large BAC clone encompassing the human OLIG2 locus that rescues motor neuron and oligodendrocyte development but not normal pattern formation in Olig2(-/-) embryos. Within the BAC clone, we identified a conserved 3.6 kb enhancer sub-region that directs reporter expression specifically in the motor neuron lineage but not oligodendrocyte lineage in vivo. Our findings indicate complex regulation of Olig2 by stage- and lineage-specific regulatory elements. They further suggest that transcriptional regulation of Olig2 is involved in segregation of pMN neuroblasts.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Linhagem da Célula/fisiologia , Neurônios Motores/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Medula Espinal/citologia , Medula Espinal/embriologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem da Célula/genética , Cromossomos Artificiais Bacterianos , Elementos Facilitadores Genéticos , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neurônios Motores/citologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Fator de Transcrição 2 de Oligodendrócitos , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Células-Tronco/citologia , Células-Tronco/fisiologiaRESUMO
Leptomeningeal glioneuronal heterotopias are a focal type of cortical dysplasia in which neural cells migrate aberrantly into superficial layers of the cerebral cortex and meninges. These heterotopias are frequently observed as microscopic abnormalities in the brains of individuals with central nervous system (CNS) malformations and epilepsy. Previous work has demonstrated that the function of Emx2, which encodes a homeodomain transcription factor, is essential for development of the cortical preplate, which gives rise to the marginal zone and subplate. However, transcriptional targets of EMX2 during CNS development are unknown. We report that leptomeningeal glioneuronal heterotopias form in Emx2(-/-) mice that are equivalent to human lesions. Additionally, we observed ectopic expression of Wnt1 in the embryonic roofplate organizer region and dorsal telencephalon. To determine the phenotypic consequences of such Wnt1 misexpression, we deleted a putative EMX2 DNA-binding site from the Wnt1 enhancer and used this to misexpress Wnt1 in the developing murine CNS. Heterotopias were detected in transgenic mice as early as 13.5 days postcoitum, consistent with a defect of preplate development during early phases of radial neuronal migration. Furthermore, we observed diffuse abnormalities of reelin- and calretinin-positive cell populations in the marginal zone and subplate similar to those observed in Emx2-null animals. Taken together, these findings indicate that EMX2 is a direct repressor of Wnt1 expression in the developing mammalian telencephalon. They further suggest that EMX2-Wnt1 interactions are essential for normal development of preplate derivatives in the mammalian cerebral cortex.
Assuntos
Coristoma/patologia , Proteínas de Homeodomínio/metabolismo , Meninges/patologia , Neuroglia , Neurônios , Proteínas Proto-Oncogênicas/metabolismo , Telencéfalo/crescimento & desenvolvimento , Proteínas de Peixe-Zebra , Animais , Sítios de Ligação , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Humanos , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/genética , Proteína Reelina , Fatores de Transcrição , Transgenes , Proteínas Wnt , Proteína Wnt1RESUMO
Olig1 and Olig2 are closely related basic helix-loop-helix (bHLH) transcription factors that are expressed in myelinating oligodendrocytes and their progenitor cells in the developing central nervous system (CNS). Olig2 is necessary for the specification of oligodendrocytes, but the biological functions of Olig1 during oligodendrocyte lineage development are poorly understood. We show here that Olig1 function in mice is required not to develop the brain but to repair it. Specifically, we demonstrate a genetic requirement for Olig1 in repairing the types of lesions that occur in patients with multiple sclerosis.