RESUMEN
Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Modelos Biológicos , Técnicas de Cultivo de Órganos , Organoides/patología , Neoplasias Pancreáticas/patología , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Páncreas/metabolismo , Páncreas/patologíaRESUMEN
Nucleotide excision repair (NER) is one of the main DNA repair pathways that protect cells against genomic damage. Disruption of this pathway can contribute to the development of cancer and accelerate aging. Mutational characteristics of NER-deficiency may reveal important diagnostic opportunities, as tumors deficient in NER are more sensitive to certain treatments. Here, we analyzed the genome-wide somatic mutational profiles of adult stem cells (ASCs) from NER-deficient Ercc1 -/Δ mice. Our results indicate that NER-deficiency increases the base substitution load twofold in liver but not in small intestinal ASCs, which coincides with the tissue-specific aging pathology observed in these mice. Moreover, NER-deficient ASCs of both tissues show an increased contribution of Signature 8 mutations, which is a mutational pattern with unknown etiology that is recurrently observed in various cancer types. The scattered genomic distribution of the base substitutions indicates that deficiency of global-genome NER (GG-NER) underlies the observed mutational consequences. In line with this, we observe increased Signature 8 mutations in a GG-NER-deficient human organoid culture, in which XPC was deleted using CRISPR-Cas9 gene-editing. Furthermore, genomes of NER-deficient breast tumors show an increased contribution of Signature 8 mutations compared with NER-proficient tumors. Elevated levels of Signature 8 mutations could therefore contribute to a predictor of NER-deficiency based on a patient's mutational profile.
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Reparación del ADN/genética , Mutación , Neoplasias/genética , Células Madre Adultas , Animales , Neoplasias de la Mama/genética , Estudios de Cohortes , Análisis Mutacional de ADN , ADN de Neoplasias , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Femenino , Humanos , Ratones , Organoides , Técnicas de Cultivo de Tejidos , Secuenciación Completa del GenomaRESUMEN
The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.
Asunto(s)
Células Madre Adultas/metabolismo , Envejecimiento/genética , Acumulación de Mutaciones , Tasa de Mutación , Especificidad de Órganos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Niño , Preescolar , Colon/metabolismo , Análisis Mutacional de ADN , Femenino , Genes Relacionados con las Neoplasias/genética , Humanos , Incidencia , Intestino Delgado/metabolismo , Hígado/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Células Madre Multipotentes/metabolismo , Neoplasias/epidemiología , Neoplasias/genética , Organoides/metabolismo , Mutación Puntual/genética , Adulto JovenRESUMEN
Intrinsic and/or acquired resistance represents one of the great challenges in targeted cancer therapy. A deeper understanding of the molecular biology of cancer has resulted in more efficient strategies, where one or multiple drugs are adopted in novel therapies to tackle resistance. This beneficial effect of using combination treatments has also been observed in colorectal cancer patients harboring the BRAF(V600E) mutation, whereby dual inhibition of BRAF(V600E) and EGFR increases antitumor activity. Notwithstanding this success, it is not clear whether this combination treatment is the only or most effective treatment to block intrinsic resistance to BRAF inhibitors. Here, we investigate molecular responses upon single and multi-target treatments, over time, using BRAF(V600E) mutant colorectal cancer cells as a model system. Through integration of transcriptomic, proteomic and phosphoproteomics data we obtain a comprehensive overview, revealing both known and novel responses. We primarily observe widespread up-regulation of receptor tyrosine kinases and metabolic pathways upon BRAF inhibition. These findings point to mechanisms by which the drug-treated cells switch energy sources and enter a quiescent-like state as a defensive response, while additionally compensating for the MAPK pathway inhibition.
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Neoplasias Colorrectales/patología , Receptores ErbB/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Biología de Sistemas/métodos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Regulación hacia Abajo/efectos de los fármacos , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Retroalimentación Fisiológica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Modelos Biológicos , Mutación/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Organs age differently, causing wide heterogeneity in multimorbidity, but underlying mechanisms are largely elusive. To investigate the basis of organ-specific ageing, we utilized progeroid repair-deficient Ercc1Δ/- mouse mutants and systematically compared at the tissue, stem cell and organoid level two organs representing ageing extremes. Ercc1Δ/- intestine shows hardly any accelerated ageing. Nevertheless, we found apoptosis and reduced numbers of intestinal stem cells (ISCs), but cell loss appears compensated by over-proliferation. ISCs retain their organoid-forming capacity, but organoids perform poorly in culture, compared with WT. Conversely, liver ages dramatically, even causing early death in Ercc1-KO mice. Apoptosis, p21, polyploidization and proliferation of various (stem) cells were prominently elevated in Ercc1Δ/- liver and stem cell populations were either largely unaffected (Sox9+), or expanding (Lgr5+), but were functionally exhausted in organoid formation and development in vitro. Paradoxically, while intestine displays less ageing, repair in WT ISCs appears inferior to liver as shown by enhanced sensitivity to various DNA-damaging agents, and lower lesion removal. Our findings reveal organ-specific anti-ageing strategies. Intestine, with short lifespan limiting time for damage accumulation and repair, favours apoptosis of damaged cells relying on ISC plasticity. Liver with low renewal rates depends more on repair pathways specifically protecting the transcribed compartment of the genome to promote sustained functionality and cell preservation. As shown before, the hematopoietic system with intermediate self-renewal mainly invokes replication-linked mechanisms, apoptosis and senescence. Hence, organs employ different genome maintenance strategies, explaining heterogeneity in organ ageing and the segmental nature of DNA-repair-deficient progerias.
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Envejecimiento , Daño del ADN , Envejecimiento/genética , Envejecimiento/metabolismo , Animales , Daño del ADN/genética , Reparación del ADN , Ratones , Organoides/metabolismo , Células Madre/metabolismoRESUMEN
Identifying the cell of origin of cancer is important to guide treatment decisions. Machine learning approaches have been proposed to classify the cell of origin based on somatic mutation profiles from solid biopsies. However, solid biopsies can cause complications and certain tumors are not accessible. Liquid biopsies are promising alternatives but their somatic mutation profile is sparse and current machine learning models fail to perform in this setting. We propose an improved method to deal with sparsity in liquid biopsy data. Firstly, data augmentation is performed on sparse data to enhance model robustness. Secondly, we employ data integration to merge information from: (i) SNV density; (ii) SNVs in driver genes and (iii) trinucleotide motifs. Our adapted method achieves an average accuracy of 0.88 and 0.65 on data where only 70% and 2% of SNVs are retained, compared to 0.83 and 0.41 with the original model, respectively. The method and results presented here open the way for application of machine learning in the detection of the cell of origin of cancer from liquid biopsy data.
RESUMEN
Levels of circulating tumor DNA (ctDNA) in liquid biopsies may serve as a sensitive biomarker for real-time, minimally-invasive tumor diagnostics and monitoring. However, detecting ctDNA is challenging, as much fewer than 5% of the cell-free DNA in the blood typically originates from the tumor. To detect lowly abundant ctDNA molecules based on somatic variants, extremely sensitive sequencing methods are required. Here, we describe a new technique, CyclomicsSeq, which is based on Oxford Nanopore sequencing of concatenated copies of a single DNA molecule. Consensus calling of the DNA copies increased the base-calling accuracy ~60×, enabling accurate detection of TP53 mutations at frequencies down to 0.02%. We demonstrate that a TP53-specific CyclomicsSeq assay can be successfully used to monitor tumor burden during treatment for head-and-neck cancer patients. CyclomicsSeq can be applied to any genomic locus and offers an accurate diagnostic liquid biopsy approach that can be implemented in clinical workflows.
RESUMEN
Inflammatory liver disease increases the risk of developing primary liver cancer. The mechanism through which liver disease induces tumorigenesis remains unclear, but is thought to occur via increased mutagenesis. Here, we performed whole-genome sequencing on clonally expanded single liver stem cells cultured as intrahepatic cholangiocyte organoids (ICOs) from patients with alcoholic cirrhosis, non-alcoholic steatohepatitis (NASH), and primary sclerosing cholangitis (PSC). Surprisingly, we find that these precancerous liver disease conditions do not result in a detectable increased accumulation of mutations, nor altered mutation types in individual liver stem cells. This finding contrasts with the mutational load and typical mutational signatures reported for liver tumors, and argues against the hypothesis that liver disease drives tumorigenesis via a direct mechanism of induced mutagenesis. Disease conditions in the liver may thus act through indirect mechanisms to drive the transition from healthy to cancerous cells, such as changes to the microenvironment that favor the outgrowth of precancerous cells.
Asunto(s)
Colangitis Esclerosante/genética , Cirrosis Hepática Alcohólica/genética , Hepatopatías/genética , Mutagénesis , Enfermedad del Hígado Graso no Alcohólico/genética , Lesiones Precancerosas/genética , Células Madre/metabolismo , Humanos , Hígado/fisiología , Organoides/metabolismoRESUMEN
Genetic changes acquired during in vitro culture pose a risk for the successful application of stem cells in regenerative medicine. To assess the genetic risks induced by culturing, we determined all mutations in individual human stem cells by whole genome sequencing. Individual pluripotent, intestinal, and liver stem cells accumulate 3.5 ± 0.5, 7.2 ± 1.1 and 8.3 ± 3.6 base substitutions per population doubling, respectively. The annual in vitro mutation accumulation rate of adult stem cells is nearly 40-fold higher than the in vivo mutation accumulation rate. Mutational signature analysis reveals that in vitro induced mutations are caused by oxidative stress. Reducing oxygen tension in culture lowers the mutational load. We use the mutation rates, spectra, and genomic distribution to model the accumulation of oncogenic mutations during typical in vitro expansion, manipulation or screening experiments using human stem cells. Our study provides empirically defined parameters to assess the mutational risk of stem cell based therapies.
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Células Madre Adultas/metabolismo , Análisis Mutacional de ADN/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Adulto , Células Madre Adultas/citología , Algoritmos , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/citología , Intestinos/citología , Hígado/citología , Hígado/metabolismo , Modelos Genéticos , Acumulación de Mutaciones , Tasa de Mutación , Medicina Regenerativa/métodos , Secuenciación Completa del Genoma/métodosRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
The development, homeostasis, and repair of intrahepatic and extrahepatic bile ducts are thought to involve distinct mechanisms including proliferation and maturation of cholangiocyte and progenitor cells. This study aimed to characterize human extrahepatic cholangiocyte organoids (ECO) using canonical Wnt-stimulated culture medium previously developed for intrahepatic cholangiocyte organoids (ICO). Paired ECO and ICO were derived from common bile duct and liver tissue, respectively. Characterization showed both organoid types were highly similar, though some differences in size and gene expression were observed. Both ECO and ICO have cholangiocyte fate differentiation capacity. However, unlike ICO, ECO lack the potential for differentiation towards a hepatocyte-like fate. Importantly, ECO derived from a cystic fibrosis patient showed no CFTR channel activity but normal chloride channel and MDR1 transporter activity. In conclusion, this study shows that ECO and ICO have distinct lineage fate and that ECO provide a competent model to study extrahepatic bile duct diseases like cystic fibrosis.
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Enfermedades de los Conductos Biliares/metabolismo , Conductos Biliares Intrahepáticos/metabolismo , Diferenciación Celular , Fibrosis Quística/metabolismo , Organoides/metabolismo , Adolescente , Enfermedades de los Conductos Biliares/patología , Conductos Biliares Intrahepáticos/patología , Fibrosis Quística/patología , Humanos , Masculino , Organoides/patologíaRESUMEN
A developing human fetus needs to balance rapid cellular expansion with maintaining genomic stability. Here, we accurately quantified and characterized somatic mutation accumulation in fetal tissues by analyzing individual stem cells from human fetal liver and intestine. Fetal mutation rates were about fivefold higher than in tissue-matched adult stem cells. The mutational landscape of fetal intestinal stem cells resembled that of adult intestinal stem cells, while the mutation spectrum of fetal liver stem cells is distinct from stem cells of the fetal intestine and the adult liver. Our analyses indicate that variation in mutational mechanisms, including oxidative stress and spontaneous deamination of methylated cytosines, contributes to the observed divergence in mutation accumulation patterns and drives genetic mosaicism in humans.
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Feto/fisiología , Mutación , Células Madre Adultas/fisiología , Feto/citología , Humanos , Intestinos/citología , Intestinos/embriología , Hígado/citología , Hígado/embriología , Tasa de Mutación , Especificidad de Órganos , Piel/citología , Piel/embriologíaRESUMEN
Characterization of mutational processes in adult stem cells (ASCs) will improve our understanding of aging-related diseases, such as cancer and organ failure, and may ultimately help prevent the development of these diseases. Here, we present a method for cataloging mutations in individual human ASCs without the necessity of using error-prone whole-genome amplification. Single ASCs are expanded in vitro into clonal organoid cultures to generate sufficient DNA for accurate whole-genome sequencing (WGS) analysis. We developed a data-analysis pipeline that identifies with high confidence somatic variants that accumulated in vivo in the original ASC. These genome-wide mutation catalogs are valuable resources for the characterization of the underlying mutational mechanisms. In addition, this protocol can be used to determine the effects of culture conditions or mutagen exposure on mutation accumulation in ASCs in vitro. Here, we describe a protocol for human liver ASCs that can be completed over a period of 3-4 months with hands-on time of â¼5 d.
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Células Madre Adultas/citología , Acumulación de Mutaciones , Mutación/genética , Organoides/citología , Secuenciación Completa del Genoma/métodos , Células Cultivadas , ADN/análisis , ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hígado/citologíaRESUMEN
Genomic rearrangements are a common cause of human congenital abnormalities. However, their origin and consequences are poorly understood. We performed molecular analysis of two patients with congenital disease who carried de novo genomic rearrangements. We found that the rearrangements in both patients hit genes that are recurrently rearranged in cancer (ETV1, FOXP1, and microRNA cluster C19MC) and drive formation of fusion genes similar to those described in cancer. Subsequent analysis of a large set of 552 de novo germline genomic rearrangements underlying congenital disorders revealed enrichment for genes rearranged in cancer and overlap with somatic cancer breakpoints. Breakpoints of common (inherited) germline structural variations also overlap with cancer breakpoints but are depleted for cancer genes. We propose that the same genomic positions are prone to genomic rearrangements in germline and soma but that timing and context of breakage determines whether developmental defects or cancer are promoted.
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Aberraciones Cromosómicas , Cromosomas Humanos/genética , Anomalías Congénitas/genética , Reordenamiento Génico , Genoma Humano , Mutación de Línea Germinal , Animales , Puntos de Rotura del Cromosoma , Proteínas de Unión al ADN/genética , Factores de Transcripción Forkhead/genética , Células HEK293 , Humanos , MicroARNs/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Pez CebraRESUMEN
Chromothripsis represents a novel phenomenon in the structural variation landscape of cancer genomes. Here, we analyze the genomes of ten patients with congenital disease who were preselected to carry complex chromosomal rearrangements with more than two breakpoints. The rearrangements displayed unanticipated complexity resembling chromothripsis. We find that eight of them contain hallmarks of multiple clustered double-stranded DNA breaks (DSBs) on one or more chromosomes. In addition, nucleotide resolution analysis of 98 breakpoint junctions indicates that break repair involves nonhomologous or microhomology-mediated end joining. We observed that these eight rearrangements are balanced or contain sporadic deletions ranging in size between a few hundred base pairs and several megabases. The two remaining complex rearrangements did not display signs of DSBs and contain duplications, indicative of rearrangement processes involving template switching. Our work provides detailed insight into the characteristics of chromothripsis and supports a role for clustered DSBs driving some constitutional chromothripsis rearrangements.