RESUMO
The integration of CRISPR/Cas9 genome editing techniques with organoid technology has revolutionized the field of tumor modeling, enabling the creation of diverse tumor models with distinct mutational profiles. This protocol details the application of CRISPR knock-ins to engineer tumor organoids with reporter cassettes, which are regulated by endogenous promoters of specific genes of interest. This approach facilitates the precise fluorescent labeling, isolation, and subsequent manipulation of targeted tumor cell subpopulations. The utilization of these knock-in reporter cassettes not only allows the visualization and purification of specific tumor cell subsets but also enables conditional cell ablation and lineage tracing studies. In this chapter, we provide a comprehensive guide for the design, construction, delivery, and validation of CRISPR/Cas9 tools tailored for knock-in reporter cassette integration into specific marker genes of interest. By following this protocol, researchers can harness the potential of engineered tumor organoids to decipher intricate tumor heterogeneity, track metastatic trajectories, and unveil novel therapeutic vulnerabilities linked to specific tumor cell subpopulations.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Técnicas de Introdução de Genes , Organoides , Organoides/metabolismo , Organoides/patologia , Humanos , Técnicas de Introdução de Genes/métodos , Edição de Genes/métodos , Animais , Neoplasias/genética , Neoplasias/patologia , Genes ReporterRESUMO
Colorectal cancer (CRC) tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing single cell RNA-sequencing (scRNA-seq) expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.
Assuntos
Neoplasias Colorretais , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Colorretais/patologia , Células-Tronco Neoplásicas/metabolismo , FenótipoRESUMO
Around 30-40% of patients with colorectal cancer (CRC) undergoing curative resection of the primary tumour will develop metastases in the subsequent years1. Therapies to prevent disease relapse remain an unmet medical need. Here we uncover the identity and features of the residual tumour cells responsible for CRC relapse. An analysis of single-cell transcriptomes of samples from patients with CRC revealed that the majority of genes associated with a poor prognosis are expressed by a unique tumour cell population that we named high-relapse cells (HRCs). We established a human-like mouse model of microsatellite-stable CRC that undergoes metastatic relapse after surgical resection of the primary tumour. Residual HRCs occult in mouse livers after primary CRC surgery gave rise to multiple cell types over time, including LGR5+ stem-like tumour cells2-4, and caused overt metastatic disease. Using Emp1 (encoding epithelial membrane protein 1) as a marker gene for HRCs, we tracked and selectively eliminated this cell population. Genetic ablation of EMP1high cells prevented metastatic recurrence and mice remained disease-free after surgery. We also found that HRC-rich micrometastases were infiltrated with T cells, yet became progressively immune-excluded during outgrowth. Treatment with neoadjuvant immunotherapy eliminated residual metastatic cells and prevented mice from relapsing after surgery. Together, our findings reveal the cell-state dynamics of residual disease in CRC and anticipate that therapies targeting HRCs may help to avoid metastatic relapse.
Assuntos
Neoplasias Colorretais , Metástase Neoplásica , Proteínas de Neoplasias , Recidiva Local de Neoplasia , Neoplasia Residual , Receptores de Superfície Celular , Animais , Humanos , Camundongos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Progressão da Doença , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/prevenção & controle , Recidiva Local de Neoplasia/terapia , Neoplasia Residual/genética , Neoplasia Residual/patologia , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Metástase Neoplásica/prevenção & controle , Metástase Neoplásica/terapia , Modelos Animais de Doenças , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/imunologia , Terapia Neoadjuvante , ImunoterapiaRESUMO
BACKGROUND: Tumor-initiating cells (TIC), also known as cancer stem cells, are considered a specific subpopulation of cells necessary for cancer initiation and metastasis; however, the mechanisms by which they acquire metastatic traits are not well understood. METHODS: LAMC2 transcriptional levels were evaluated using publicly available transcriptome data sets, and LAMC2 immunohistochemistry was performed using a tissue microarray composed of PDAC and normal pancreas tissues. Silencing and tracing of LAMC2 was performed using lentiviral shRNA constructs and CRISPR/Cas9-mediated homologous recombination, respectively. The contribution of LAMC2 to PDAC tumorigenicity was explored in vitro by tumor cell invasion, migration, sphere-forming and organoids assays, and in vivo by tumor growth and metastatic assays. mRNA sequencing was performed to identify key cellular pathways upregulated in LAMC2 expressing cells. Metastatic spreading induced by LAMC2- expressing cells was blocked by pharmacological inhibition of transforming growth factor beta (TGF-ß) signaling. RESULTS: We report a LAMC2-expressing cell population, which is endowed with enhanced self-renewal capacity, and is sufficient for tumor initiation and differentiation, and drives metastasis. mRNA profiling of these cells indicates a prominent squamous signature, and differentially activated pathways critical for tumor growth and metastasis, including deregulation of the TGF-ß signaling pathway. Treatment with Vactosertib, a new small molecule inhibitor of the TGF-ß type I receptor (activin receptor-like kinase-5, ALK5), completely abrogated lung metastasis, primarily originating from LAMC2-expressing cells. CONCLUSIONS: We have identified a highly metastatic subpopulation of TICs marked by LAMC2. Strategies aimed at targeting the LAMC2 population may be effective in reducing tumor aggressiveness in PDAC patients. Our results prompt further study of this TIC population in pancreatic cancer and exploration as a potential therapeutic target and/or biomarker.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Carcinoma Ductal Pancreático/patologia , Receptor do Fator de Crescimento Transformador beta Tipo I , RNA Interferente Pequeno , Neoplasias Pancreáticas/patologia , Células-Tronco Neoplásicas/metabolismo , Fator de Crescimento Transformador beta , RNA Mensageiro , Receptores de Ativinas , Movimento Celular/genética , Linhagem Celular Tumoral , Laminina/genética , Laminina/metabolismo , Neoplasias PancreáticasRESUMO
Colorectal cancer (CRC) patient-derived organoids predict responses to chemotherapy. Here we used them to investigate relapse after treatment. Patient-derived organoids expand from highly proliferative LGR5+ tumor cells; however, we discovered that lack of optimal growth conditions specifies a latent LGR5+ cell state. This cell population expressed the gene MEX3A, is chemoresistant and regenerated the organoid culture after treatment. In CRC mouse models, Mex3a+ cells contributed marginally to metastatic outgrowth; however, after chemotherapy, Mex3a+ cells produced large cell clones that regenerated the disease. Lineage-tracing analysis showed that persister Mex3a+ cells downregulate the WNT/stem cell gene program immediately after chemotherapy and adopt a transient state reminiscent to that of YAP+ fetal intestinal progenitors. In contrast, Mex3a-deficient cells differentiated toward a goblet cell-like phenotype and were unable to resist chemotherapy. Our findings reveal that adaptation of cancer stem cells to suboptimal niche environments protects them from chemotherapy and identify a candidate cell of origin of relapse after treatment in CRC.
Assuntos
Neoplasias Colorretais , Organoides , Animais , Diferenciação Celular , Neoplasias Colorretais/tratamento farmacológico , Camundongos , Células-Tronco Neoplásicas , RecidivaRESUMO
Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.
Assuntos
Anticorpos Biespecíficos , Neoplasias Epiteliais e Glandulares , Anticorpos Biespecíficos/farmacologia , Receptores ErbB/metabolismo , Humanos , Imidazóis , Neoplasias Epiteliais e Glandulares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Organoides , Pirazinas , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Colorectal cancers (CRCs) are composed of an amalgam of cells with distinct genotypes and phenotypes. Here, we reveal a previously unappreciated heterogeneity in the biosynthetic capacities of CRC cells. We discover that the majority of ribosomal DNA transcription and protein synthesis in CRCs occurs in a limited subset of tumor cells that localize in defined niches. The rest of the tumor cells undergo an irreversible loss of their biosynthetic capacities as a consequence of differentiation. Cancer cells within the biosynthetic domains are characterized by elevated levels of the RNA polymerase I subunit A (POLR1A). Genetic ablation of POLR1A-high cell population imposes an irreversible growth arrest on CRCs. We show that elevated biosynthesis defines stemness in both LGR5+ and LGR5- tumor cells. Therefore, a common architecture in CRCs is a simple cell hierarchy based on the differential capacity to transcribe ribosomal DNA and synthesize proteins.
Assuntos
Neoplasias Colorretais , Células-Tronco Neoplásicas , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , DNA Ribossômico , Humanos , Receptores Acoplados a Proteínas GRESUMO
The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of identifying or tracking tumor cell populations in an intact environment. To overcome this limitation, we devised a strategy based on editing the genomes of patient-derived tumor organoids using CRISPR/Cas9 technology to integrate reporter cassettes at desired marker genes. As proof of concept, we engineered human colorectal cancer (CRC) organoids that carry EGFP and lineage-tracing cassettes knocked in the LGR5 locus. Analysis of LGR5-EGFP+ cells isolated from organoid-derived xenografts demonstrated that these cells express a gene program similar to that of normal intestinal stem cells and that they propagate the disease to recipient mice very efficiently. Lineage-tracing experiments showed that LGR5+ CRC cells self-renew and generate progeny over long time periods that undergo differentiation toward mucosecreting- and absorptive-like phenotypes. These genetic experiments confirm that human CRCs adopt a hierarchical organization reminiscent of that of the normal colonic epithelium. The strategy described herein may have broad applications to study cell heterogeneity in human tumors.
Assuntos
Técnicas de Cultura de Células/métodos , Neoplasias Colorretais/fisiopatologia , Células-Tronco Neoplásicas/fisiologia , Organoides , Animais , Diferenciação Celular , Proliferação de Células , Feminino , Edição de Genes/métodos , Técnicas de Introdução de Genes , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Xenoenxertos , Humanos , Camundongos SCID , Receptores Acoplados a Proteínas G/genética , Coloração e Rotulagem/métodosRESUMO
The contribution of somatic mutations to metastasis of colorectal cancers is currently unknown. To find mutations involved in the colorectal cancer metastatic process, we performed deep mutational analysis of 676 genes in 107 stages II to IV primary colorectal cancer, of which half had metastasized. The mutation prevalence in the ephrin (EPH) family of tyrosine kinase receptors was 10-fold higher in primary tumors of metastatic colorectal than in nonmetastatic cases and preferentially occurred in stage III and IV tumors. Mutational analyses in situ confirmed expression of mutant EPH receptors. To enable functional studies of EPHB1 mutations, we demonstrated that DLD-1 colorectal cancer cells expressing EPHB1 form aggregates upon coculture with ephrin B1 expressing cells. When mutations in the fibronectin type III and kinase domains of EPHB1 were compared with wild-type EPHB1 in DLD-1 colorectal cancer cells, they decreased ephrin B1-induced compartmentalization. These observations provide a mechanistic link between EPHB receptor mutations and metastasis in colorectal cancer. Cancer Res; 77(7); 1730-40. ©2017 AACR.
Assuntos
Neoplasias Colorretais/patologia , Mutação , Metástase Neoplásica , Receptor EphB1/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Domínio de Fibronectina Tipo III/genética , Humanos , Estadiamento de Neoplasias , Proteínas Tirosina Quinases/genéticaRESUMO
Recent molecular classifications of colorectal cancer (CRC) based on global gene expression profiles have defined subtypes displaying resistance to therapy and poor prognosis. Upon evaluation of these classification systems, we discovered that their predictive power arises from genes expressed by stromal cells rather than epithelial tumor cells. Bioinformatic and immunohistochemical analyses identify stromal markers that associate robustly with disease relapse across the various classifications. Functional studies indicate that cancer-associated fibroblasts (CAFs) increase the frequency of tumor-initiating cells, an effect that is dramatically enhanced by transforming growth factor (TGF)-ß signaling. Likewise, we find that all poor-prognosis CRC subtypes share a gene program induced by TGF-ß in tumor stromal cells. Using patient-derived tumor organoids and xenografts, we show that the use of TGF-ß signaling inhibitors to block the cross-talk between cancer cells and the microenvironment halts disease progression.
Assuntos
Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Fibroblastos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Análise por Conglomerados , Neoplasias Colorretais/classificação , Neoplasias Colorretais/patologia , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Camundongos , Camundongos Nus , Análise em Microsséries , Invasividade Neoplásica , Metástase Neoplásica , Células-Tronco Neoplásicas/patologia , Prognóstico , Células Estromais/metabolismo , Células Estromais/patologia , TranscriptomaRESUMO
Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations leading to the initiation of colorectal cancer (CRC). Here we screen for genes required for maintaining the tumour stem cell phenotype and identify the zinc-finger transcription factor GATA6 as a key regulator of the WNT and BMP pathways in CRC. GATA6 directly drives the expression of LGR5 in adenoma stem cells whereas it restricts BMP signalling to differentiated tumour cells. Genetic deletion of Gata6 from mouse colon adenomas increases the levels of BMP factors, which signal to block self-renewal of tumour stem cells. In human tumours, GATA6 competes with ß-catenin/TCF4 for binding to a distal regulatory region of the BMP4 locus that has been linked to increased susceptibility to development of CRC. Hence, GATA6 creates an environment permissive for CRC initiation by lowering the threshold of BMP signalling required for tumour stem cell expansion.
Assuntos
Adenoma , Receptores de Proteínas Morfogenéticas Ósseas/genética , Neoplasias Colorretais/fisiopatologia , Fator de Transcrição GATA6/metabolismo , Regulação Neoplásica da Expressão Gênica , Células-Tronco/citologia , Células-Tronco/metabolismo , Adenoma/patologia , Animais , Antineoplásicos/farmacologia , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Proliferação de Células , Feminino , Imunofluorescência , Fator de Transcrição GATA6/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Células-Tronco/efeitos dos fármacos , Proteínas Wnt/metabolismoRESUMO
Since the initial discovery that OCT4, SOX2, KLF4, and c-MYC overexpression sufficed for the induction of pluripotency in somatic cells, methodologies replacing the original factors have enhanced our understanding of the reprogramming process. However, unlike in mouse, OCT4 has not been replaced successfully during reprogramming of human cells. Here we report on a strategy to accomplish this replacement. Through a combination of transcriptome and bioinformatic analysis we have identified factors previously characterized as being lineage specifiers that are able to replace OCT4 and SOX2 in the reprogramming of human fibroblasts. Our results show that it is possible to replace OCT4 and SOX2 simultaneously with alternative lineage specifiers in the reprogramming of human cells. At a broader level, they also support a model in which counteracting lineage specification networks underlies the induction of pluripotency.
Assuntos
Desdiferenciação Celular , Fibroblastos/fisiologia , Fator de Transcrição GATA3/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/fisiologia , Desdiferenciação Celular/genética , Linhagem da Célula , Células Cultivadas , Biologia Computacional/métodos , Fator de Transcrição GATA3/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Regeneração Tecidual Guiada , Humanos , Fator 4 Semelhante a Kruppel , Fator 3 de Transcrição de Octâmero/genética , RNA Interferente Pequeno/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transgenes/genéticaRESUMO
The tubular epithelium of the kidney is susceptible to injury from a number of different causes, including inflammatory and immune disorders, oxidative stress, and nephrotoxins, among others. Primary renal epithelial cells remain one of the few tools for studying the biochemical and physiological characteristics of the renal tubular system. Nevertheless, differentiated primary cells are not suitable for recapitulation of disease properties that might arise during embryonic kidney formation and further maturation. Thus, cellular systems resembling kidney characteristics are in urgent need to model disease as well as to establish reliable drug-testing platforms. Induced pluripotent stem cells (iPSCs) bear the capacity to differentiate into every cell lineage comprising the adult organism. Thus, iPSCs bring the possibility for recapitulating embryonic development by directed differentiation into specific lineages. iPSC differentiation ultimately allows for both disease modeling in vitro and the production of cellular products with potential for regenerative medicine. Here, we describe the rapid, reproducible, and highly efficient generation of iPSCs derived from endogenous kidney tubular renal epithelial cells with only two transcriptional factors, OCT4 and SOX2. Kidney-derived iPSCs may provide a reliable cellular platform for the development of kidney differentiation protocols allowing drug discovery studies and the study of kidney pathology.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-Idade , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genéticaRESUMO
The formation and maintenance of complex organs requires segregation of distinct cell populations into defined territories (that is, cell sorting) and the establishment of boundaries between them. Here we have investigated the mechanism by which Eph/ephrin signalling controls the compartmentalization of cells in epithelial tissues. We show that EphB/ephrin-B signalling in epithelial cells regulates the formation of E-cadherin-based adhesions. EphB receptors interact with E-cadherin and with the metalloproteinase ADAM10 at sites of adhesion and their activation induces shedding of E-cadherin by ADAM10 at interfaces with ephrin-B1-expressing cells. This process results in asymmetric localization of E-cadherin and, as a consequence, in differences in cell affinity between EphB-positive and ephrin-B-positive cells. Furthermore, genetic inhibition of ADAM10 activity in the intestine of mice results in a lack of compartmentalization of Paneth cells within the crypt stem cell niche, a defect that phenocopies that of EphB3-null mice. These results provide important insights into the regulation of cell migration in the intestinal epithelium and may help in the understanding of the nature of the cell sorting process in other epithelial tissues where Eph-ephrin interactions play a central role.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Caderinas/metabolismo , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Receptores da Família Eph/metabolismo , Transdução de Sinais , Proteínas ADAM/genética , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/genética , Animais , Western Blotting , Caderinas/genética , Adesão Celular , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Mucosa Intestinal/metabolismo , Intestinos/citologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência , Celulas de Paneth/metabolismo , Ligação Proteica , Interferência de RNA , Receptor EphB1/genética , Receptor EphB1/metabolismo , Receptor EphB3/genética , Receptor EphB3/metabolismo , Receptores da Família Eph/genética , Nicho de Células-TroncoRESUMO
The genes encoding tyrosine kinase receptors EphB2 and EphB3 are beta-catenin and Tcf4 target genes in colorectal cancer (CRC) and in normal intestinal cells. In the intestinal epithelium, EphB signaling controls the positioning of cell types along the crypt-villus axis. In CRC, EphB activity suppresses tumor progression beyond the earliest stages. Here we show that EphB receptors compartmentalize the expansion of CRC cells through a mechanism dependent on E-cadherin-mediated adhesion. We demonstrate that EphB-mediated compartmentalization restricts the spreading of EphB-expressing tumor cells into ephrin-B1-positive territories in vitro and in vivo. Our results indicate that CRC cells must silence EphB expression to avoid repulsive interactions imposed by normal ephrin-B1-expressing intestinal cells at the onset of tumorigenesis.