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Direct investigation of the early cellular changes induced by metastatic cells within the surrounding tissue remains a challenge. Here we present a system in which metastatic cancer cells release a cell-penetrating fluorescent protein, which is taken up by neighbouring cells and enables spatial identification of the local metastatic cellular environment. Using this system, tissue cells with low representation in the metastatic niche can be identified and characterized within the bulk tissue. To highlight its potential, we applied this strategy to study the cellular environment of metastatic breast cancer cells in the lung. We report the presence of cancer-associated parenchymal cells, which exhibit stem-cell-like features, expression of lung progenitor markers, multi-lineage differentiation potential and self-renewal activity. In ex vivo assays, lung epithelial cells acquire a cancer-associated parenchymal-cell-like phenotype when co-cultured with cancer cells and support their growth. These results highlight the potential of this method as a platform for new discoveries.
Assuntos
Linhagem da Célula , Rastreamento de Células/métodos , Metástase Neoplásica/patologia , Células-Tronco Neoplásicas/patologia , Tecido Parenquimatoso/patologia , Coloração e Rotulagem/métodos , Nicho de Células-Tronco , Microambiente Tumoral , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Diferenciação Celular , Técnicas de Cocultura , Células Epiteliais/patologia , Feminino , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Metástase Neoplásica/imunologia , Neutrófilos/patologia , Organoides/patologia , Nicho de Células-Tronco/imunologia , Microambiente Tumoral/imunologia , Proteína Vermelha FluorescenteRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The heterochromatin barrier must be overcome to generate induced pluripotent stem cells and cell fusion-mediated reprogrammed hybrids. Here, we show that the absence of T-cell factor 3 (Tcf3), a repressor of ß-catenin target genes, strikingly and rapidly enhances the efficiency of neural precursor cell (NPC) reprogramming. Remarkably, Tcf3(-/-) ES cells showed a genome-wide increase in AcH3 and decrease in H3K9me3 and can reprogram NPCs after fusion greatly. In addition, during reprogramming of NPCs into induced pluripotent stem cells, the silencing of Tcf3 increased AcH3 and decreased the number of H3K9me3-positive heterochromatin foci early and long before reactivation of the endogenous stem cell genes. In conclusion, our data suggest that Tcf3 functions as a repressor of the reprogramming potential of somatic cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Reprogramação Celular/fisiologia , Epigênese Genética/fisiologia , Deleção de Genes , Células-Tronco Pluripotentes Induzidas/fisiologia , Neurônios/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular , Reprogramação Celular/genética , Imunoprecipitação da Cromatina , Epigênese Genética/genética , Citometria de Fluxo , Imunofluorescência , Vetores Genéticos/genética , Immunoblotting , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Retroviridae , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Isolation of live cells from the tumor microenvironment (TME) has represented a challenge, particularly from metastatic nodules that need to be identified within the entire metastatic tissue. Cherry-niche, an in vivo labelling technique, allows the isolation of all the different cell populations in the TME without needing to visually locate the metastatic cancer cell colonies. Therefore, neighboring TME cells can be isolated even from the early stages of cancer cell seeding and colonization in the metastatic tissue. Here, we show how to use Cherry-niche to identify and isolate neutrophils from the lung metastatic niche. We also provide examples of downstream analyses to characterize freshly isolated neutrophils ex vivo, such as Giemsa staining, reactive oxygen species (ROS) detection, and phagocytosis assays. Similar strategies can be used to isolate other immune and non-immune cells from the metastatic TME.
Assuntos
Pulmão , Microambiente Tumoral , Pulmão/patologia , Neutrófilos/patologia , Linhagem Celular TumoralRESUMO
A key step for metastatic outgrowth involves the generation of a deeply altered microenvironment (niche) that supports the malignant behavior of cancer cells. The complexity of the metastatic niche has posed a significant challenge in elucidating the underlying programs driving its origin. Here, by focusing on early stages of breast cancer metastasis to the lung in mice, we describe a cancer-dependent chromatin remodeling and activation of developmental programs in alveolar type 2 (AT2) cells within the niche. We show that metastatic cells can prime AT2 cells into a reprogrammed multilineage state. In turn, this cancer-induced reprogramming of AT2 cells promoted stem-like features in cancer cells and enhanced their initiation capacity. In conclusion, we propose the concept of "reflected stemness" as an early phenomenon during metastatic niche initiation, wherein metastatic cells reprogram the local tissue into a stem-like state that enhances intrinsic cancer-initiating potential, creating a positive feedback loop where tumorigenic programs are amplified.
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Cancer cells thrive when embedded in a fine-tuned cellular and extracellular environment or tumour microenvironment (TME). There is a general understanding of a co-evolution between cancer cells and their surrounding TME, pointing at a functional connection between cancer cells characteristics and the perturbations induced in their surrounding tissue. However, it has never been formally proven whether this functional connection needs to be set from the start or if aggressive cancer cells always dominate their microenvironmental any point in time. This would require a dedicated experimental setting where malignant cells are challenged to grow in a different TME from the one they would naturally create. Here we generated an experimental setting where we transiently perturb the secretory profile of aggressive breast cancer cells without affecting their intrinsic growth ability, which led to the initial establishment of an atypical TME. Interestingly, even if initially tumours are formed, this atypical TME evolves to impair long term in vivo cancer growth. Using a combination of in vivo transcriptomics, protein arrays and in vitro co-cultures, we found that the atypical TME culminates in the infiltration of macrophages with STAT1high activity. These macrophages show strong anti-tumoural functions which reduce long-term tumour growth, despite lacking canonical M1 markers. Importantly, gene signatures of the mesenchymal compartment of the TME, as well as the anti-tumoural macrophages, show striking prognostic power that correlates with less aggressive human breast cancers.
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Neoplasias da Mama , Microambiente Tumoral , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Humanos , Macrófagos/patologiaRESUMO
Radiotherapy is one of the most effective approaches to achieve tumor control in cancer patients, although healthy tissue injury due to off-target radiation exposure can occur. In this study, we used a model of acute radiation injury to the lung, in the context of cancer metastasis, to understand the biological link between tissue damage and cancer progression. We exposed healthy mouse lung tissue to radiation before the induction of metastasis and observed a strong enhancement of cancer cell growth. We found that locally activated neutrophils were key drivers of the tumor-supportive preconditioning of the lung microenvironment, governed by enhanced regenerative Notch signaling. Importantly, these tissue perturbations endowed arriving cancer cells with an augmented stemness phenotype. By preventing neutrophil-dependent Notch activation, via blocking degranulation, we were able to significantly offset the radiation-enhanced metastases. This work highlights a pro-tumorigenic activity of neutrophils, which is likely linked to their tissue regenerative functions.
Assuntos
Neoplasias Pulmonares , Exposição à Radiação , Animais , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Camundongos , Ativação de Neutrófilo , Neutrófilos/patologia , Microambiente TumoralRESUMO
The maintenance of genome stability relies on coordinated control of origin activation and replication fork progression. How the interplay between these processes influences human genetic disease and cancer remains incompletely characterized. Here we show that mouse cells featuring Polε instability exhibit impaired genome-wide activation of DNA replication origins, in an origin-location-independent manner. Strikingly, Trp53 ablation in primary Polε hypomorphic cells increased Polε levels and origin activation and reduced DNA damage in a transcription-dependent manner. Transcriptome analysis of primary Trp53 knockout cells revealed that the TRP53-CDKN1A/P21 axis maintains appropriate levels of replication factors and CDK activity during unchallenged S phase. Loss of this control mechanism deregulates origin activation and perturbs genome-wide replication fork progression. Thus, while our data support an impaired origin activation model for genetic diseases affecting CMG formation, we propose that loss of the TRP53-CDKN1A/P21 tumor suppressor axis induces inappropriate origin activation and deregulates genome-wide fork progression.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21 , DNA Polimerase II , Replicação do DNA , Proteínas de Ligação a Poli-ADP-Ribose , Origem de Replicação , Proteína Supressora de Tumor p53 , Animais , Proteínas de Ciclo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Dano ao DNA/genética , DNA Polimerase II/genética , Replicação do DNA/genética , Camundongos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Fase S , Proteína Supressora de Tumor p53/genéticaRESUMO
Understanding cell-cell interactions is critical in most, if not all, research fields in biology. Nevertheless, studying intercellular crosstalk in vivo remains a relevant challenge, due mainly to the difficulty in spatially locating the surroundings of particular cells in the tissue. Cherry-niche is a powerful new method that enables cells expressing a fluorescent protein to label their surrounding cells, facilitating their specific isolation from the whole tissue as live cells. We previously applied Cherry-niche in cancer research to study the tumor microenvironment (TME) in metastasis. Here we describe how to generate cancer cells with the ability to label their neighboring cells (within the tumor niche) by transferring a liposoluble fluorescent protein. Live niche cells can be isolated and compared with cells distant from the tumor bulk, using a variety of ex vivo approaches. As previously shown, this system has the potential to identify novel components in the TME and improve our understanding of their local interactions. Importantly, Cherry-niche can also be applied to study potential cell-cell interactions due to in vivo proximity in research fields beyond cancer. This protocol takes 2-3 weeks to generate the labeling cells and 1-2 weeks to test their labeling ability.
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Comunicação Celular/fisiologia , Imuno-Histoquímica/métodos , Corantes Fluorescentes/química , Humanos , Neoplasias/imunologia , Neoplasias/patologia , Microambiente Tumoral/imunologia , Microambiente Tumoral/fisiologiaRESUMO
The bone-marrow (BM) niche is the spatial environment composed by a network of multiple stromal components regulating adult hematopoiesis. We use multi-omics and computational tools to analyze multiple BM environmental compartments and decipher their mutual interactions in the context of acute myeloid leukemia (AML) xenografts. Under homeostatic conditions, we find a considerable overlap between niche populations identified using current markers. Our analysis defines eight functional clusters of genes informing on the cellular identity and function of the different subpopulations and pointing at specific stromal interrelationships. We describe how these transcriptomic profiles change during human AML development and, by using a proximity-based molecular approach, we identify early disease onset deregulated genes in the mesenchymal compartment. Finally, we analyze the BM proteomic secretome in the presence of AML and integrate it with the transcriptome to predict signaling nodes involved in niche alteration in AML.
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Células da Medula Óssea/metabolismo , Leucemia Mieloide Aguda/genética , Proteômica/métodos , Animais , Humanos , Camundongos , Microambiente TumoralRESUMO
Metastases are a major cause of cancer-related death and despite the fact that they have been focus of intense research over the last two decades, effective therapies for patients with distant secondary lesions are still very limited. In addition, in some tumor types metastases can grow years after the patients have been declared clinically cured, indicating that disseminated cancer cells (DCCs) persist undetected for years, even decades in a quiescent state. Clinical and experimental data highlight the importance of the immune system in shaping the fitness and behaviour of DCCs. Here, we review mechanisms of survival, quiescence and outgrowth of DCCs with a special focus on immune-regulation and we highlight the latest cutting-edge techniques for modelling the biology of DCCs in vitro and for studying the metastatic niche in vivo. We believe that a wide dissemination of those techniques will boost scientific findings towards new therapies to defeat metastatic relapses in cancer patients.
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Converting carcinomas in benign oncocytomas has been suggested as a potential anti-cancer strategy. One of the oncocytoma hallmarks is the lack of respiratory complex I (CI). Here we use genetic ablation of this enzyme to induce indolence in two cancer types, and show this is reversed by allowing the stabilization of Hypoxia Inducible Factor-1 alpha (HIF-1α). We further show that on the long run CI-deficient tumors re-adapt to their inability to respond to hypoxia, concordantly with the persistence of human oncocytomas. We demonstrate that CI-deficient tumors survive and carry out angiogenesis, despite their inability to stabilize HIF-1α. Such adaptive response is mediated by tumor associated macrophages, whose blockage improves the effect of CI ablation. Additionally, the simultaneous pharmacological inhibition of CI function through metformin and macrophage infiltration through PLX-3397 impairs tumor growth in vivo in a synergistic manner, setting the basis for an efficient combinatorial adjuvant therapy in clinical trials.
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Adenoma Oxífilo/tratamento farmacológico , Adenoma Oxífilo/genética , Aminopiridinas/farmacologia , Antineoplásicos/farmacologia , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Metformina/farmacologia , Pirróis/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Drosophila , Feminino , Técnicas de Inativação de Genes , Células HCT116 , Humanos , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Camundongos Nus , NADH Desidrogenase/genética , Neovascularização Patológica/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
During metastatic colonization, tumor cells must establish a favorable microenvironment or niche that will sustain their growth. However, both the temporal and molecular details of this process remain poorly understood. Here, we found that metastatic initiating cells (MICs) exhibit a high capacity for lung fibroblast activation as a result of Thrombospondin 2 (THBS2) expression. Importantly, inhibiting the mesenchymal phenotype of MICs by blocking the epithelial-to-mesenchymal transition (EMT)-associated kinase AXL reduces THBS2 secretion, niche-activating ability, and, consequently, metastatic competence. Subsequently, disseminated metastatic cells revert to an AXL-negative, more epithelial phenotype to proliferate and decrease the phosphorylation levels of TGF-ß-dependent SMAD2-3 in favor of BMP/SMAD1-5 signaling. Remarkably, newly activated fibroblasts promote this transition. In summary, our data reveal a crosstalk between cancer cells and their microenvironment whereby the EMT status initially triggers and then is regulated by niche activation during metastatic colonization.
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Células-Tronco Neoplásicas/metabolismo , Trombospondinas/metabolismo , Animais , Benzocicloeptenos/toxicidade , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Antígeno CD24/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Camundongos Transgênicos , Metástase Neoplásica , Células-Tronco Neoplásicas/citologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Proteínas Smad/genética , Proteínas Smad/metabolismo , Trombospondinas/antagonistas & inibidores , Trombospondinas/genética , Fator de Crescimento Transformador beta/metabolismo , Transplante Heterólogo , Triazóis/toxicidade , Receptor Tirosina Quinase AxlRESUMO
Tumor metastasis, the cause of more than 90% of cancer cell mortality, is a multistep process by which tumor cells disseminate from their primary site via local invasion and intravasation into blood or lymphatic vessels and reach secondary distant sites, where they survive and reinitiate tumor growth. Activation of a developmental program called the epithelial-to-mesenchymal transition (EMT) has been shown to be a very efficient strategy adopted by epithelial cancer cells to promote local invasion and dissemination at distant organs. Remarkably, the activation of EMT programs in epithelial cells correlates with the appearance of stemness. This finding suggests that the EMT process also drives the initial cancer cell colonization at distant sites. However, recent studies support the concept that its reverse program, a mesenchymal-to-epithelial transition, is required for efficient metastatic colonization and that EMT is not necessarily associated with stemness. This review analyzes the conflicting experimental evidence linking epithelial plasticity to stemness in the light of an "EMT gradient model," according to which the outcome of EMT program activation in epithelial cells would be bimodal: coupled to stemness during initial activation, but when forced to reach an advanced mesenchymal status, it would become incompatible with stem cell abilities.
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Divisão Celular , Transição Epitelial-Mesenquimal , Metástase Neoplásica , Neoplasias/patologia , Humanos , Células-Tronco Neoplásicas/patologiaRESUMO
Cyclic activation of the Wnt/ß-catenin signaling pathway controls cell fusion-mediated somatic cell reprogramming. TCFs belong to a family of transcription factors that, in complex with ß-catenin, bind and transcriptionally regulate Wnt target genes. Here, we show that Wnt/ß-catenin signaling needs to be off during the early reprogramming phases of mouse embryonic fibroblasts (MEFs) into iPSCs. In MEFs undergoing reprogramming, senescence genes are repressed and mesenchymal-to-epithelial transition is favored. This is correlated with a repressive activity of TCF1, which contributes to the silencing of Wnt/ß-catenin signaling at the onset of reprogramming. In contrast, the Wnt pathway needs to be active in the late reprogramming phases to achieve successful reprogramming. In conclusion, continued activation or inhibition of the Wnt/ß-catenin signaling pathway is detrimental to the reprogramming of MEFs; instead, temporal perturbation of the pathway is essential for efficient reprogramming, and the "Wnt-off" state can be considered an early reprogramming marker.
Assuntos
Fator 1-alfa Nuclear de Hepatócito/metabolismo , Via de Sinalização Wnt , Animais , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular , Reprogramação Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Transição Epitelial-Mesenquimal , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 1-alfa Nuclear de Hepatócito/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Wnt/ß-catenin canonical pathway has very important roles both in the regulation of self-renewal and lineage differentiation of pluripotent stem cells, as well as in the reprogramming of somatic cells. In the absence of Wnt stimulation, the target genes of the pathway are repressed or down-regulated. Conversely, when ß-catenin is stabilized there is an accumulation of cytoplasmic and nuclear ß-catenin that allows up-regulation of its target genes. However, the molecular mechanisms downstream of ß-catenin that regulate self-renewal of embryonic stem cells remain unclear. It has been proposed that ß-catenin acts through members of the T-cell factor (TCF) family. Recent studies have confirmed the role of Tcf3 (Tcf7l1) as a transcriptional repressor in embryonic stem cells, and they have also proposed a new mechanistic view as to how activation of the Wnt/ß-catenin pathway can overcome this repression.