RESUMO
Mismatch repair (MMR)-deficient cancer evolves through the stepwise erosion of coding homopolymers in target genes. Curiously, the MMR genes MutS homolog 6 (MSH6) and MutS homolog 3 (MSH3) also contain coding homopolymers, and these are frequent mutational targets in MMR-deficient cancers. The impact of incremental MMR mutations on MMR-deficient cancer evolution is unknown. Here we show that microsatellite instability modulates DNA repair by toggling hypermutable mononucleotide homopolymer runs in MSH6 and MSH3 through stochastic frameshift switching. Spontaneous mutation and reversion modulate subclonal mutation rate, mutation bias and HLA and neoantigen diversity. Patient-derived organoids corroborate these observations and show that MMR homopolymer sequences drift back into reading frame in the absence of immune selection, suggesting a fitness cost of elevated mutation rates. Combined experimental and simulation studies demonstrate that subclonal immune selection favors incremental MMR mutations. Overall, our data demonstrate that MMR-deficient colorectal cancers fuel intratumor heterogeneity by adapting subclonal mutation rate and diversity to immune selection.
Assuntos
Neoplasias Colorretais , Reparo de Erro de Pareamento de DNA , Instabilidade de Microssatélites , Humanos , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/genética , Mutação , Proteína 3 Homóloga a MutS/genética , Taxa de Mutação , Mutação da Fase de Leitura/genéticaRESUMO
In response to excessive DNA damage, human cells can activate p53 to induce apoptosis. Cells lacking p53 can still undergo apoptosis upon DNA damage, yet the responsible pathways are unknown. We observed that p53-independent apoptosis in response to DNA damage coincided with translation inhibition, which was characterized by ribosome stalling on rare leucine-encoding UUA codons and globally curtailed translation initiation. A genetic screen identified the transfer RNAse SLFN11 and the kinase GCN2 as factors required for UUA stalling and global translation inhibition, respectively. Stalled ribosomes activated a ribotoxic stress signal conveyed by the ribosome sensor ZAKα to the apoptosis machinery. These results provide an explanation for the frequent inactivation of SLFN11 in chemotherapy-unresponsive tumors and highlight ribosome stalling as a signaling event affecting cell fate in response to DNA damage.
Assuntos
Apoptose , Dano ao DNA , Biossíntese de Proteínas , Ribossomos , Proteína Supressora de Tumor p53 , Humanos , Linhagem Celular Tumoral , Códon/genética , Leucina/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Ribossomos/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismoRESUMO
Oncogene-induced senescence is a phenomenon in which aberrant oncogene expression causes non-transformed cells to enter a non-proliferative state. Cells undergoing oncogenic induction display phenotypic heterogeneity, with some cells senescing and others remaining proliferative. The causes of heterogeneity remain unclear. We studied the sources of heterogeneity in the responses of human epithelial cells to oncogenic BRAFV600E expression. We found that a narrow expression range of BRAFV600E generated a wide range of activities of its downstream effector ERK. In population-level and single-cell assays, ERK activity displayed a non-monotonic relationship to proliferation, with intermediate ERK activities leading to maximal proliferation. We profiled gene expression across a range of ERK activities over time and characterized four distinct ERK response classes, which we propose act in concert to generate the ERK-proliferation response. Altogether, our studies map the input-output relationships between ERK activity and proliferation, elucidating how heterogeneity can be generated during oncogene induction.
Assuntos
Oncogenes , Proteínas Proto-Oncogênicas B-raf , Humanos , Linhagem Celular Tumoral , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismoRESUMO
Patient-derived organoids (PDOs) are widely heralded as a drug-screening platform to develop new anti-cancer therapies. Here, we use a drug-repurposing library to screen PDOs of colorectal cancer (CRC) to identify hidden vulnerabilities within therapy-induced phenotypes. Using a microscopy-based screen that accurately scores drug-induced cell killing, we have tested 414 putative anti-cancer drugs for their ability to switch the EGFRi/MEKi-induced cytostatic phenotype toward cytotoxicity. A majority of validated hits (9/37) are microtubule-targeting agents that are commonly used in clinical oncology, such as taxanes and vinca-alkaloids. One of these drugs, vinorelbine, is consistently effective across a panel of >25 different CRC PDOs, independent of RAS mutational status. Unlike vinorelbine alone, its combination with EGFR/MEK inhibition induces apoptosis at all stages of the cell cycle and shows tolerability and effective anti-tumor activity in vivo, setting the basis for a clinical trial to treat patients with metastatic RAS-mutant CRC.
Assuntos
Antineoplásicos , Neoplasias do Colo , Neoplasias Colorretais , Humanos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Vinorelbina/farmacologia , Vinorelbina/uso terapêutico , Reposicionamento de Medicamentos , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Organoides/metabolismoRESUMO
The morphology and functionality of the epithelial lining differ along the intestinal tract, but tissue renewal at all sites is driven by stem cells at the base of crypts1-3. Whether stem cell numbers and behaviour vary at different sites is unknown. Here we show using intravital microscopy that, despite similarities in the number and distribution of proliferative cells with an Lgr5 signature in mice, small intestinal crypts contain twice as many effective stem cells as large intestinal crypts. We find that, although passively displaced by a conveyor-belt-like upward movement, small intestinal cells positioned away from the crypt base can function as long-term effective stem cells owing to Wnt-dependent retrograde cellular movement. By contrast, the near absence of retrograde movement in the large intestine restricts cell repositioning, leading to a reduction in effective stem cell number. Moreover, after suppression of the retrograde movement in the small intestine, the number of effective stem cells is reduced, and the rate of monoclonal conversion of crypts is accelerated. Together, these results show that the number of effective stem cells is determined by active retrograde movement, revealing a new channel of stem cell regulation that can be experimentally and pharmacologically manipulated.
Assuntos
Contagem de Células , Movimento Celular , Intestinos , Células-Tronco , Animais , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Intestinos/citologia , Camundongos , Receptores Acoplados a Proteínas G , Células-Tronco/citologia , Proteínas WntRESUMO
Micrometastases of colorectal cancer can remain dormant for years prior to the formation of actively growing, clinically detectable lesions (i.e., colonization). A better understanding of this step in the metastatic cascade could help improve metastasis prevention and treatment. Here we analyzed liver specimens of patients with colorectal cancer and monitored real-time metastasis formation in mouse livers using intravital microscopy to reveal that micrometastatic lesions are devoid of cancer stem cells (CSC). However, lesions that grow into overt metastases demonstrated appearance of de novo CSCs through cellular plasticity at a multicellular stage. Clonal outgrowth of patient-derived colorectal cancer organoids phenocopied the cellular and transcriptomic changes observed during in vivo metastasis formation. First, formation of mature CSCs occurred at a multicellular stage and promoted growth. Conversely, failure of immature CSCs to generate more differentiated cells arrested growth, implying that cellular heterogeneity is required for continuous growth. Second, early-stage YAP activity was required for the survival of organoid-forming cells. However, subsequent attenuation of early-stage YAP activity was essential to allow for the formation of cell type heterogeneity, while persistent YAP signaling locked micro-organoids in a cellularly homogenous and growth-stalled state. Analysis of metastasis formation in mouse livers using single-cell RNA sequencing confirmed the transient presence of early-stage YAP activity, followed by emergence of CSC and non-CSC phenotypes, irrespective of the initial phenotype of the metastatic cell of origin. Thus, establishment of cellular heterogeneity after an initial YAP-controlled outgrowth phase marks the transition to continuously growing macrometastases. SIGNIFICANCE: Characterization of the cell type dynamics, composition, and transcriptome of early colorectal cancer liver metastases reveals that failure to establish cellular heterogeneity through YAP-controlled epithelial self-organization prohibits the outgrowth of micrometastases. See related commentary by LeBleu, p. 1870.
Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , Animais , Neoplasias Colorretais/patologia , Humanos , Neoplasias Hepáticas/metabolismo , Camundongos , Micrometástase de Neoplasia/patologia , Células-Tronco Neoplásicas/patologiaRESUMO
CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.
Assuntos
DNA/genética , Desoxirribonuclease I/metabolismo , Loci Gênicos , Organoides/metabolismo , Reparo de DNA por Recombinação , Coloração e Rotulagem/métodos , Alelos , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Colo/citologia , Colo/metabolismo , DNA/metabolismo , Reparo do DNA por Junção de Extremidades , Desoxirribonuclease I/genética , Eletroporação/métodos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Técnicas de Introdução de Genes , Vetores Genéticos , Genoma Humano , Heterozigoto , Humanos , Organoides/citologiaRESUMO
Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq-a protocol that integrates live-cell imaging of tumor organoid outgrowth and whole-genome sequencing of each imaged cell to reconstruct evolving tumor cell karyotypes across consecutive cell generations. Using patient-derived colorectal cancer organoids and fresh tumor biopsies, we demonstrate that karyotype alterations of varying complexity are prevalent and can arise within a few cell generations. Sub-chromosomal acentric fragments were prone to replication and collective missegregation across consecutive cell divisions. In contrast, gross genome-wide karyotype alterations were generated in a single erroneous cell division, providing support that aneuploid tumor genomes can evolve via punctuated evolution. Mapping the temporal dynamics and patterns of karyotype diversification in cancer enables reconstructions of evolutionary paths to malignant fitness.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Análise de Célula Única/métodos , Proliferação de Células/genética , Cromatina/genética , Cromossomos Humanos , Dosagem de Genes , Humanos , Cariótipo , Cariotipagem , Microscopia Confocal , Mitose , Organoides/crescimento & desenvolvimento , Organoides/patologia , Fuso Acromático/genéticaRESUMO
Survival rates of cancer patients vary widely within and between malignancies. While genetic aberrations are at the root of all cancers, individual genomic features cannot explain these distinct disease outcomes. In contrast, intra-tumour heterogeneity (ITH) has the potential to elucidate pan-cancer survival rates and the biology that drives cancer prognosis. Unfortunately, a comprehensive and effective framework to measure ITH across cancers is missing. Here, we introduce a scalable measure of chromosomal copy number heterogeneity (CNH) that predicts patient survival across cancers. We show that the level of ITH can be derived from a single-sample copy number profile. Using gene-expression data and live cell imaging we demonstrate that ongoing chromosomal instability underlies the observed heterogeneity. Analysing 11,534 primary cancer samples from 37 different malignancies, we find that copy number heterogeneity can be accurately deduced and predicts cancer survival across tissues of origin and stages of disease. Our results provide a unifying molecular explanation for the different survival rates observed between cancer types.
Assuntos
Variações do Número de Cópias de DNA , Heterogeneidade Genética , Modelos Genéticos , Neoplasias/mortalidade , Microambiente Tumoral/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Simulação por Computador , Conjuntos de Dados como Assunto , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genômica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasias/genética , Neoplasias/patologia , Prognóstico , Intervalo Livre de Progressão , Medição de Risco/métodos , Taxa de Sobrevida , Adulto JovemRESUMO
Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Mutação , Organoides/metabolismo , Organoides/patologia , Análise de Célula ÚnicaRESUMO
Patient-derived organoids maintain functional and phenotypic characteristics of the original tissue such as cell-type diversity. Here, we provide protocols on how to label intestinal (cancer) stem cells by integrating the stem cell ASCL2 reporter (STAR) into human and mouse genomes via two different strategies: (1) lentiviral transduction or (2) transposon-based integration. Organoid technology, in combination with the user-friendly nature of STAR, will facilitate basic research in human and mouse adult stem cell biology. For complete details on the use and execution of this protocol, please refer to Oost et al. (2018).
Assuntos
Organoides/metabolismo , Coloração e Rotulagem/métodos , Células-Tronco Adultas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Humanos , Mucosa Intestinal/diagnóstico por imagem , Intestinos/citologia , Camundongos , Organoides/crescimento & desenvolvimento , Células-Tronco/metabolismoRESUMO
Damage to the intestinal stem cell niche can result from mechanical stress, infections, chronic inflammation or cytotoxic therapies. Progenitor cells can compensate for insults to the stem cell population through dedifferentiation. The microenvironment modulates this regenerative response by influencing the activity of signaling pathways, including Wnt, Notch, and YAP/TAZ. For instance, mesenchymal cells and immune cells become more abundant after damage and secrete signaling molecules that promote the regenerative process. Furthermore, regeneration is influenced by the nutritional state, microbiome, and extracellular matrix. Here, we review how all these components cooperate to restore epithelial homeostasis in the intestine after injury.
Assuntos
Desdiferenciação Celular/genética , Intestinos/crescimento & desenvolvimento , Regeneração/genética , Células-Tronco/citologia , Aciltransferases , Proteínas de Ciclo Celular/genética , Linhagem da Célula/genética , Microambiente Celular/genética , Humanos , Intestinos/citologia , Receptores Notch/genética , Fatores de Transcrição/genética , Via de Sinalização Wnt/genéticaRESUMO
Colorectal cancer stem cells (CSCs) express Lgr5 and display extensive stem cell-like multipotency and self-renewal and are thought to seed metastatic disease. Here, we used a mouse model of colorectal cancer (CRC) and human tumor xenografts to investigate the cell of origin of metastases. We found that most disseminated CRC cells in circulation were Lgr5- and formed distant metastases in which Lgr5+ CSCs appeared. This plasticity occurred independently of stemness-inducing microenvironmental factors and was indispensable for outgrowth, but not establishment, of metastases. Together, these findings show that most colorectal cancer metastases are seeded by Lgr5- cells, which display intrinsic capacity to become CSCs in a niche-independent manner and can restore epithelial hierarchies in metastatic tumors.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Biomarcadores Tumorais , Humanos , Células-Tronco Neoplásicas , Receptores Acoplados a Proteínas GRESUMO
RAS and BRAF proteins are frequently mutated in colorectal cancer (CRC) and have been associated with therapy resistance in metastatic CRC patients. RAS isoforms are considered to act as redundant entities in physiological and pathological settings. However, there is compelling evidence that mutant variants of RAS and BRAF have different oncogenic potentials and therapeutic outcomes. In this review we describe similarities and differences between various RAS and BRAF oncogenes in CRC development, histology, and therapy resistance. In addition, we discuss the potential of patient-derived tumor organoids for personalized therapy, as well as CRC modeling using genome editing in preclinical model systems to study similarities and discrepancies between the effects of oncogenic MAPK pathway mutations on tumor growth and drug response.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/genética , Organoides/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas ras/genética , Animais , Antineoplásicos/uso terapêutico , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Fusion genes can be oncogenic drivers in a variety of cancer types and represent potential targets for targeted therapy. The BRAF gene is frequently involved in oncogenic gene fusions, with fusion frequencies of 0.2%-3% throughout different cancers. However, BRAF fusions rarely occur in the same gene configuration, potentially challenging personalized therapy design. In particular, the impact of the wide variety of fusion partners on the oncogenic role of BRAF during tumor growth and drug response is unknown. Here, we used patient-derived colorectal cancer organoids to functionally characterize and cross-compare BRAF fusions containing various partner genes (AGAP3, DLG1, and TRIM24) with respect to cellular behavior, downstream signaling activation, and response to targeted therapies. We demonstrate that 5' fusion partners mainly promote canonical oncogenic BRAF activity by replacing the auto-inhibitory N-terminal region. In addition, the 5' partner of BRAF fusions influences their subcellular localization and intracellular signaling capacity, revealing distinct subsets of affected signaling pathways and altered gene expression. Presence of the different BRAF fusions resulted in varying sensitivities to combinatorial inhibition of MEK and the EGF receptor family. However, all BRAF fusions conveyed resistance to targeted monotherapy against the EGF receptor family, suggesting that BRAF fusions should be screened alongside other MAPK pathway alterations to identify patients with metastatic colorectal cancer to exclude from anti-EGFR-targeted treatment. IMPLICATIONS: Although intracellular signaling and sensitivity to targeted therapies of BRAF fusion genes are influenced by their 5' fusion partner, we show that all investigated BRAF fusions confer resistance to clinically relevant EGFR inhibition.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Diferenciação Celular/fisiologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Células HEK293 , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Terapia de Alvo Molecular , Fusão Oncogênica , Organoides , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland.
Assuntos
Técnicas de Cultura de Células/métodos , Organoides/crescimento & desenvolvimento , Venenos de Serpentes/metabolismo , Células-Tronco Adultas/metabolismo , Animais , Cobras Corais/metabolismo , Perfilação da Expressão Gênica/métodos , Organoides/metabolismo , Glândulas Salivares/metabolismo , Venenos de Serpentes/genética , Serpentes/genética , Serpentes/crescimento & desenvolvimento , Células-Tronco/metabolismo , Toxinas Biológicas/genética , Transcriptoma/genéticaRESUMO
Chromosome segregation errors cause aneuploidy and genomic heterogeneity, which are hallmarks of cancer in humans. A persistent high frequency of these errors (chromosomal instability (CIN)) is predicted to profoundly impact tumor evolution and therapy response. It is unknown, however, how prevalent CIN is in human tumors. Using three-dimensional live-cell imaging of patient-derived tumor organoids (tumor PDOs), we show that CIN is widespread in colorectal carcinomas regardless of background genetic alterations, including microsatellite instability. Cell-fate tracking showed that, although mitotic errors are frequently followed by cell death, some tumor PDOs are largely insensitive to mitotic errors. Single-cell karyotype sequencing confirmed heterogeneity of copy number alterations in tumor PDOs and showed that monoclonal lines evolved novel karyotypes over time in vitro. We conclude that ongoing CIN is common in colorectal cancer organoids, and propose that CIN levels and the tolerance for mitotic errors shape aneuploidy landscapes and karyotype heterogeneity.
Assuntos
Instabilidade Cromossômica , Neoplasias Colorretais/genética , Aneuploidia , Linhagem Celular Tumoral , Segregação de Cromossomos , Neoplasias Colorretais/patologia , Variações do Número de Cópias de DNA , Humanos , Imageamento Tridimensional , Cariótipo , Cariotipagem , Instabilidade de Microssatélites , Mitose/genética , Mutação , Organoides/patologia , Análise de Célula ÚnicaRESUMO
Coordinated induction, but also repression, of genes are key to normal differentiation. Although the role of lineage-specific transcription regulators has been studied extensively, their functional integration with chromatin remodelers, one of the key enzymatic machineries that control chromatin accessibility, remains ill-defined. Here we investigate the role of Mi-2ß, a SNF-2-like nucleosome remodeler and key component of the nucleosome remodeling and histone deacetylase (NuRD) complex in early B cells. Inactivation of Mi-2ß arrested differentiation at the large pre-B-cell stage and caused derepression of cell adhesion and cell migration signaling factors by increasing chromatin access at poised enhancers and chromosome architectural elements. Mi-2ß also supported IL-7R signaling, survival, and proliferation by repressing negative effectors of this pathway. Importantly, overexpression of Bcl2, a mitochondrial prosurvival gene and target of IL-7R signaling, partly rescued the differentiation block caused by Mi-2ß loss. Mi-2ß stably associated with chromatin sites that harbor binding motifs for IKAROS and EBF1 and physically associated with these transcription factors both on and off chromatin. Notably, Mi-2ß shared loss-of-function cellular and molecular phenotypes with IKAROS and EBF1, albeit in a distinct fashion. Thus, the nucleosome remodeler Mi-2ß promotes pre-B-cell differentiation by providing repression capabilities to distinct lineage-specific transcription factor-based regulatory networks.
Assuntos
Linfócitos B/citologia , Diferenciação Celular/genética , Cromatina/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Animais , Linhagem da Célula , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Camundongos , Fatores de TranscriçãoRESUMO
In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.
Assuntos
Imageamento Tridimensional/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Organoides/ultraestrutura , Fixação de Tecidos/métodos , Animais , Mama/ultraestrutura , Colo/ultraestrutura , Feminino , Humanos , Imuno-Histoquímica/métodos , Rim/ultraestrutura , Fígado/ultraestrutura , CamundongosRESUMO
Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing all main subtypes of OC. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra- and interpatient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug-screening assays and capture different tumor subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug-sensitivity assays. Taken together, this demonstrates their potential application for research and personalized medicine.