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1.
Cell ; 179(5): 1207-1221.e22, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31730858

RESUMO

Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.


Assuntos
Replicação do DNA/genética , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Célula Única , Aneuploidia , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Forma Celular , Sobrevivência Celular , Cromossomos Humanos/genética , Células Clonais , Elementos de DNA Transponíveis/genética , Diploide , Feminino , Genótipo , Humanos , Masculino , Camundongos , Mutação/genética , Filogenia , Polimorfismo de Nucleotídeo Único/genética
2.
Cell ; 167(1): 260-274.e22, 2016 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-27641504

RESUMO

The inter- and intra-tumor heterogeneity of breast cancer needs to be adequately captured in pre-clinical models. We have created a large collection of breast cancer patient-derived tumor xenografts (PDTXs), in which the morphological and molecular characteristics of the originating tumor are preserved through passaging in the mouse. An integrated platform combining in vivo maintenance of these PDTXs along with short-term cultures of PDTX-derived tumor cells (PDTCs) was optimized. Remarkably, the intra-tumor genomic clonal architecture present in the originating breast cancers was mostly preserved upon serial passaging in xenografts and in short-term cultured PDTCs. We assessed drug responses in PDTCs on a high-throughput platform and validated several ex vivo responses in vivo. The biobank represents a powerful resource for pre-clinical breast cancer pharmacogenomic studies (http://caldaslab.cruk.cam.ac.uk/bcape), including identification of biomarkers of response or resistance.


Assuntos
Bancos de Espécimes Biológicos , Neoplasias da Mama , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Biomarcadores Farmacológicos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Testes Farmacogenômicos , Células Tumorais Cultivadas
3.
Nature ; 612(7938): 106-115, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36289342

RESUMO

How cell-to-cell copy number alterations that underpin genomic instability1 in human cancers drive genomic and phenotypic variation, and consequently the evolution of cancer2, remains understudied. Here, by applying scaled single-cell whole-genome sequencing3 to wild-type, TP53-deficient and TP53-deficient;BRCA1-deficient or TP53-deficient;BRCA2-deficient mammary epithelial cells (13,818 genomes), and to primary triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC) cells (22,057 genomes), we identify three distinct 'foreground' mutational patterns that are defined by cell-to-cell structural variation. Cell- and clone-specific high-level amplifications, parallel haplotype-specific copy number alterations and copy number segment length variation (serrate structural variations) had measurable phenotypic and evolutionary consequences. In TNBC and HGSC, clone-specific high-level amplifications in known oncogenes were highly prevalent in tumours bearing fold-back inversions, relative to tumours with homologous recombination deficiency, and were associated with increased clone-to-clone phenotypic variation. Parallel haplotype-specific alterations were also commonly observed, leading to phylogenetic evolutionary diversity and clone-specific mono-allelic expression. Serrate variants were increased in tumours with fold-back inversions and were highly correlated with increased genomic diversity of cellular populations. Together, our findings show that cell-to-cell structural variation contributes to the origins of phenotypic and evolutionary diversity in TNBC and HGSC, and provide insight into the genomic and mutational states of individual cancer cells.


Assuntos
Genômica , Mutação , Neoplasias Ovarianas , Análise de Célula Única , Neoplasias de Mama Triplo Negativas , Feminino , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Filogenia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
4.
Nature ; 595(7868): 585-590, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34163070

RESUMO

Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models1-7. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model8,9 to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.


Assuntos
Variações do Número de Cópias de DNA , Resistencia a Medicamentos Antineoplásicos , Neoplasias de Mama Triplo Negativas/genética , Animais , Linhagem Celular Tumoral , Cisplatino/farmacologia , Células Clonais/patologia , Feminino , Aptidão Genética , Humanos , Camundongos , Modelos Estatísticos , Transplante de Neoplasias , Proteína Supressora de Tumor p53/genética , Sequenciamento Completo do Genoma
5.
Nature ; 518(7539): 422-6, 2015 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-25470049

RESUMO

Human cancers, including breast cancers, comprise clones differing in mutation content. Clones evolve dynamically in space and time following principles of Darwinian evolution, underpinning important emergent features such as drug resistance and metastasis. Human breast cancer xenoengraftment is used as a means of capturing and studying tumour biology, and breast tumour xenografts are generally assumed to be reasonable models of the originating tumours. However, the consequences and reproducibility of engraftment and propagation on the genomic clonal architecture of tumours have not been systematically examined at single-cell resolution. Here we show, using deep-genome and single-cell sequencing methods, the clonal dynamics of initial engraftment and subsequent serial propagation of primary and metastatic human breast cancers in immunodeficient mice. In all 15 cases examined, clonal selection on engraftment was observed in both primary and metastatic breast tumours, varying in degree from extreme selective engraftment of minor (<5% of starting population) clones to moderate, polyclonal engraftment. Furthermore, ongoing clonal dynamics during serial passaging is a feature of tumours experiencing modest initial selection. Through single-cell sequencing, we show that major mutation clusters estimated from tumour population sequencing relate predictably to the most abundant clonal genotypes, even in clonally complex and rapidly evolving cases. Finally, we show that similar clonal expansion patterns can emerge in independent grafts of the same starting tumour population, indicating that genomic aberrations can be reproducible determinants of evolutionary trajectories. Our results show that measurement of genomically defined clonal population dynamics will be highly informative for functional studies using patient-derived breast cancer xenoengraftment.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Células Clonais/metabolismo , Células Clonais/patologia , Genoma Humano/genética , Análise de Célula Única , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Neoplasias da Mama/secundário , Análise Mutacional de DNA , Genômica , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Transplante de Neoplasias , Fatores de Tempo , Transplante Heterólogo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
6.
Nat Methods ; 14(2): 167-173, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28068316

RESUMO

Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing library preparation methods are expensive, require sample preamplification and introduce coverage bias. Here we describe direct library preparation (DLP), a robust, scalable, and high-fidelity method that uses nanoliter-volume transposition reactions for single-cell whole-genome library preparation without preamplification. We examined 782 cells from cell lines and triple-negative breast xenograft tumors. Low-depth sequencing, compared with existing methods, revealed greater coverage uniformity and more reliable detection of copy-number alterations. Using phylogenetic analysis, we found minor xenograft subpopulations that were undetectable by bulk sequencing, as well as dynamic clonal expansion and diversification between passages. Merging single-cell genomes in silico, we generated 'bulk-equivalent' genomes with high depth and uniform coverage. Thus, low-depth sequencing of DLP libraries may provide an attractive replacement for conventional bulk sequencing methods, permitting analysis of copy number at the cell level and of other genomic variants at the population level.


Assuntos
Genômica/métodos , Análise de Célula Única/métodos , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Biblioteca Gênica , Humanos , Dispositivos Lab-On-A-Chip , Camundongos SCID , Filogenia , Análise de Célula Única/instrumentação , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Proc Natl Acad Sci U S A ; 111(21): 7789-94, 2014 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-24821780

RESUMO

Mechanisms that control the levels and activities of reactive oxygen species (ROS) in normal human mammary cells are poorly understood. We show that purified normal human basal mammary epithelial cells maintain low levels of ROS primarily by a glutathione-dependent but inefficient antioxidant mechanism that uses mitochondrial glutathione peroxidase 2. In contrast, the matching purified luminal progenitor cells contain higher levels of ROS, multiple glutathione-independent antioxidants and oxidative nucleotide damage-controlling proteins and consume O2 at a higher rate. The luminal progenitor cells are more resistant to glutathione depletion than the basal cells, including those with in vivo and in vitro proliferation and differentiation activity. The luminal progenitors also are more resistant to H2O2 or ionizing radiation. Importantly, even freshly isolated "steady-state" normal luminal progenitors show elevated levels of unrepaired oxidative DNA damage. Distinct ROS control mechanisms operating in different subsets of normal human mammary cells could have differentiation state-specific functions and long-term consequences.


Assuntos
Células Epiteliais/classificação , Células Epiteliais/metabolismo , Glutationa/metabolismo , Glândulas Mamárias Humanas/citologia , Estresse Oxidativo/fisiologia , Western Blotting , Dano ao DNA/fisiologia , Citometria de Fluxo , Humanos , Consumo de Oxigênio/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Células-Tronco/metabolismo
8.
PLoS Biol ; 11(8): e1001630, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23966837

RESUMO

Many normal adult tissues contain rare stem cells with extensive self-maintaining regenerative potential. During development, the stem cells of the hematopoietic and neural systems undergo intrinsically specified changes in their self-renewal potential. In the mouse, mammary stem cells with transplantable regenerative activity are first detectable a few days before birth. They share some phenotypic properties with their adult counterparts but are enriched in a subpopulation that displays a distinct gene expression profile. Here we show that fetal mammary epithelial cells have a greater direct and inducible growth potential than their adult counterparts. The latter feature is revealed in a novel culture system that enables large numbers of in vitro clonogenic progenitors as well as mammary stem cells with serially transplantable activity to be produced within 7 days from single fetal or adult input cells. We further show that these responses are highly dependent on novel factors produced by fibroblasts. These findings provide new avenues for elucidating mechanisms that regulate normal mammary epithelial stem cell properties at the single-cell level, how these change during development, and how their perturbation may contribute to transformation.


Assuntos
Células Epiteliais/citologia , Glândulas Mamárias Animais/citologia , Células 3T3 , Animais , Células Epiteliais/fisiologia , Feminino , Imuno-Histoquímica , Glândulas Mamárias Animais/fisiologia , Camundongos
9.
Breast Cancer Res ; 17: 4, 2015 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-25572802

RESUMO

INTRODUCTION: The extracellular signals regulating mammary epithelial cell growth are of relevance to understanding the pathophysiology of mammary epithelia, yet they remain poorly characterized. In this study, we applied an unbiased approach to understanding the functional role of signalling molecules in several models of normal physiological growth and translated these results to the biological understanding of breast cancer subtypes. METHODS: We developed and utilized a cytogenetically normal clonal line of hTERT immortalized human mammary epithelial cells in a fibroblast-enhanced co-culture assay to conduct a genome-wide small interfering RNA (siRNA) screen for evaluation of the functional effect of silencing each gene. Our selected endpoint was inhibition of growth. In rigorous postscreen validation processes, including quantitative RT-PCR, to ensure on-target silencing, deconvolution of pooled siRNAs and independent confirmation of effects with lentiviral short-hairpin RNA constructs, we identified a subset of genes required for mammary epithelial cell growth. Using three-dimensional Matrigel growth and differentiation assays and primary human mammary epithelial cell colony assays, we confirmed that these growth effects were not limited to the 184-hTERT cell line. We utilized the METABRIC dataset of 1,998 breast cancer patients to evaluate both the differential expression of these genes across breast cancer subtypes and their prognostic significance. RESULTS: We identified 47 genes that are critically important for fibroblast-enhanced mammary epithelial cell growth. This group was enriched for several axonal guidance molecules and G protein-coupled receptors, as well as for the endothelin receptor PROCR. The majority of genes (43 of 47) identified in two dimensions were also required for three-dimensional growth, with HSD17B2, SNN and PROCR showing greater than tenfold reductions in acinar formation. Several genes, including PROCR and the neuronal pathfinding molecules EFNA4 and NTN1, were also required for proper differentiation and polarization in three-dimensional cultures. The 47 genes identified showed a significant nonrandom enrichment for differential expression among 10 molecular subtypes of breast cancer sampled from 1,998 patients. CD79A, SERPINH1, KCNJ5 and TMEM14C exhibited breast cancer subtype-independent overall survival differences. CONCLUSION: Diverse transmembrane signals are required for mammary epithelial cell growth in two-dimensional and three-dimensional conditions. Strikingly, we define novel roles for axonal pathfinding receptors and ligands and the endothelin receptor in both growth and differentiation.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Interferência de RNA , Transdução de Sinais , Adulto , Animais , Neoplasias da Mama/patologia , Comunicação Celular , Diferenciação Celular , Linhagem Celular Transformada , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Análise por Conglomerados , Técnicas de Cocultura , Feminino , Fibroblastos/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla/métodos , Ensaios de Triagem em Larga Escala , Humanos , Cariótipo , Camundongos , RNA Interferente Pequeno/genética , Esferoides Celulares , Telomerase/genética , Células Tumorais Cultivadas , Adulto Jovem
10.
Oncologist ; 19(6): 623-30, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24807916

RESUMO

Extraordinary advancements in sequencing technology have made what was once a decade-long multi-institutional endeavor into a methodology with the potential for practical use in a clinical setting. We therefore set out to examine the clinical value of next-generation sequencing by enrolling patients with incurable or ambiguous tumors into the Personalized OncoGenomics initiative at the British Columbia Cancer Agency whereby whole genome and transcriptome analyses of tumor/normal tissue pairs are completed with the ultimate goal of directing therapeutics. First, we established that the sequencing, analysis, and communication with oncologists could be completed in less than 5 weeks. Second, we found that cancer diagnostics is an area that can greatly benefit from the comprehensiveness of a whole genome analysis. Here, we present a scenario in which a metastasized sphenoid mass, which was initially thought of as an undifferentiated squamous cell carcinoma, was rediagnosed as an SMARCB1-negative rhabdoid tumor based on the newly acquired finding of homozygous SMARCB1 deletion. The new diagnosis led to a change in chemotherapy and a complete nodal response in the patient. This study also provides additional insight into the mutational landscape of an adult SMARCB1-negative tumor that has not been explored at a whole genome and transcriptome level.


Assuntos
Carcinoma de Células Escamosas/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Tumor Rabdoide/genética , Fatores de Transcrição/genética , Adulto , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/enzimologia , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Genoma Humano , Humanos , Masculino , Tumor Rabdoide/tratamento farmacológico , Tumor Rabdoide/patologia , Proteína SMARCB1
11.
Clin Chem ; 60(1): 122-33, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24298072

RESUMO

BACKGROUND: Triple-negative breast cancers (TNBC) do not represent a single disease subgroup and are often aggressive breast cancers with poor prognoses. Unlike estrogen/progesterone receptor and HER2 (human epidermal growth factor receptor 2) breast cancers, which are responsive to targeted treatments, there is no effective targeted therapy for TNBC, although approximately 50% of patients respond to conventional chemotherapies, including taxanes, anthracyclines, cyclophosphamide, and platinum salts. CONTENT: Genomic studies have helped clarify some of the possible disease groupings that make up TNBC. We discuss the findings, including copy number-transcriptome analysis, whole genome sequencing, and exome sequencing, in terms of the biological properties and phenotypes that make up the constellation of TNBC. The relationships between subgroups defined by transcriptome and genome analysis are discussed. SUMMARY: TNBC is not a uniform molecular or disease entity but a constellation of variably well-defined biological properties whose relationship to each other is not understood. There is good support for the existence of a basal expression subtype, p53 mutated, high-genomic instability subtype of TNBC. This should be considered a distinct TNBC subtype. Other subtypes with variable degrees of supporting evidence exist within the nonbasal/p53wt (wild-type p53) TNBC, including a group of TNBC with PI3K (phosphoinositide 3-kinase) pathway activation that have better overall prognosis than the basal TNBC. Consistent molecular phenotyping of TNBC by whole genome sequencing, transcriptomics, and functional studies with patient-derived tumor xenograft models will be essential components in clinical and biological studies as means of resolving this heterogeneity.


Assuntos
Genômica , Neoplasias de Mama Triplo Negativas/genética , Feminino , Perfilação da Expressão Gênica , Genes p53/genética , Humanos
12.
Stem Cells ; 30(2): 344-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22131125

RESUMO

Elevated aldehyde dehydrogenase (ALDH) expression/activity has been identified as an important biomarker of primitive cells in various normal and malignant human tissues. Here we examined the level and type of ALDH expression and activity in different subsets of phenotypically and functionally defined normal human mammary cells. We find that the most primitive human mammary stem and progenitor cell types with bilineage differentiation potential show low ALDH activity but undergo a marked, selective, and transient upregulation of ALDH activity at the point of commitment to the luminal lineage. This mirrors a corresponding change in transcripts and protein levels of ALDH1A3, an enzyme involved in retinoic acid synthesis and the most highly expressed ALDH gene in normal human mammary tissue. In contrast, ALDH1A1 is expressed at low levels in all mammary epithelial cells. These findings raise interesting questions about the reported association of ALDH activity with breast cancer stem cells and breast cancer prognosis.


Assuntos
Células-Tronco Adultas/enzimologia , Aldeído Desidrogenase/metabolismo , Glândulas Mamárias Humanas/citologia , Adulto , Aldeído Desidrogenase/genética , Família Aldeído Desidrogenase 1 , Aldeído Oxirredutases , Biomarcadores/metabolismo , Células Cultivadas , Células Epiteliais/enzimologia , Feminino , Humanos , Glândulas Mamárias Humanas/enzimologia , Retinal Desidrogenase , Células Estromais/enzimologia , Transcrição Gênica , Adulto Jovem
13.
Breast Cancer Res ; 14(5): R134, 2012 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-23088371

RESUMO

INTRODUCTION: The organisation of the mammary epithelial hierarchy is poorly understood. Our hypothesis is that the luminal cell compartment is more complex than initially described, and that an understanding of the developmental relationships within this lineage will help in understanding the cellular context in which breast tumours occur. METHODS: We used fluorescence-activated cell sorting along with in vitro and in vivo functional assays to examine the growth and differentiation properties of distinct subsets of human and mouse mammary epithelial cells. We also examined how loss of steroid hormones influenced these populations in vivo. Gene expression profiles were also obtained for all the purified cell populations and correlated to those obtained from breast tumours. RESULTS: The luminal cell compartment of the mouse mammary gland can be resolved into nonclonogenic oestrogen receptor-positive (ER+) luminal cells, ER+ luminal progenitors and oestrogen receptor-negative (ER-) luminal progenitors. The ER+ luminal progenitors are unique in regard to cell survival, as they are relatively insensitive to loss of oestrogen and progesterone when compared with the other types of mammary epithelial cells. Analysis of normal human breast tissue reveals a similar hierarchical organisation composed of nonclonogenic luminal cells, and relatively differentiated (EpCAM+CD49f+ALDH-) and undifferentiated (EpCAM+CD49f+ALDH+) luminal progenitors. In addition, approximately one-quarter of human breast samples examined contained an additional population that had a distinct luminal progenitor phenotype, characterised by low expression of ERBB3 and low proliferative potential. Parent-progeny relationship experiments demonstrated that all luminal progenitor populations in both species are highly plastic and, at low frequencies, can generate progeny representing all mammary cell types. The ER- luminal progenitors in the mouse and the ALDH+ luminal progenitors in the human appear to be analogous populations since they both have gene signatures that are associated with alveolar differentiation and resemble those obtained from basal-like breast tumours. CONCLUSION: The luminal cell compartment in the mammary epithelium is more heterogeneous than initially perceived since progenitors of varying levels of luminal cell differentiation and proliferative capacities can be identified. An understanding of these cells will be essential for understanding the origins and the cellular context of human breast tumours.


Assuntos
Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , Fenótipo , Animais , Antígenos de Superfície/metabolismo , Diferenciação Celular , Ensaio de Unidades Formadoras de Colônias , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Feminino , Humanos , Imunofenotipagem , Camundongos , Receptores de Estrogênio/metabolismo , Células-Tronco/metabolismo
14.
Nature ; 439(7079): 993-7, 2006 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-16395311

RESUMO

Elucidation of the cellular and molecular mechanisms that maintain mammary epithelial tissue integrity is of broad interest and paramount to the design of more effective treatments for breast cancer. Evidence from both in vitro and in vivo experiments suggests that mammary cell differentiation is a hierarchical process originating in an uncommitted stem cell with self-renewal potential. However, analysis of the properties and regulation of mammary stem cells has been limited by a lack of methods for their prospective isolation. Here we report the use of multi-parameter cell sorting and limiting dilution transplant analysis to demonstrate the purification of a rare subset of adult mouse mammary cells that are able individually to regenerate an entire mammary gland within 6 weeks in vivo while simultaneously executing up to ten symmetrical self-renewal divisions. These mammary stem cells are phenotypically distinct from and give rise to mammary epithelial progenitor cells that produce adherent colonies in vitro. The mammary stem cells are also a rapidly cycling population in the normal adult and have molecular features indicative of a basal position in the mammary epithelium.


Assuntos
Separação Celular/métodos , Células Epiteliais/citologia , Glândulas Mamárias Animais/citologia , Células-Tronco/citologia , Tecido Adiposo/citologia , Tecido Adiposo/transplante , Animais , Animais Congênicos , Diferenciação Celular , Proliferação de Células , Corantes/metabolismo , Feminino , Glândulas Mamárias Animais/anatomia & histologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Abstinência Sexual
15.
Nat Commun ; 13(1): 4534, 2022 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-35927228

RESUMO

Assessing tumour gene fitness in physiologically-relevant model systems is challenging due to biological features of in vivo tumour regeneration, including extreme variations in single cell lineage progeny. Here we develop a reproducible, quantitative approach to pooled genetic perturbation in patient-derived xenografts (PDXs), by encoding single cell output from transplanted CRISPR-transduced cells in combination with a Bayesian hierarchical model. We apply this to 181 PDX transplants from 21 breast cancer patients. We show that uncertainty in fitness estimates depends critically on the number of transplant cell clones and the variability in clone sizes. We use a pathway-directed allelic series to characterize Notch signaling, and quantify TP53 / MDM2 drug-gene conditional fitness in outlier patients. We show that fitness outlier identification can be mirrored by pharmacological perturbation. Overall, we demonstrate that the gene fitness landscape in breast PDXs is dominated by inter-patient differences.


Assuntos
Neoplasias da Mama , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Animais , Teorema de Bayes , Neoplasias da Mama/genética , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Genome Biol ; 20(1): 210, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31623682

RESUMO

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) is a powerful tool for studying complex biological systems, such as tumor heterogeneity and tissue microenvironments. However, the sources of technical and biological variation in primary solid tumor tissues and patient-derived mouse xenografts for scRNA-seq are not well understood. RESULTS: We use low temperature (6 °C) protease and collagenase (37 °C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNA-seq dataset comprising 155,165 cells from patient cancer tissues, patient-derived breast cancer xenografts, and cancer cell lines. We observe substantial variation in standard quality control metrics of cell viability across conditions and tissues. From the contrast between tissue protease dissociation at 37 °C or 6 °C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, including FOS and JUN, induced by collagenase (37 °C), which are minimized by dissociation with a cold active protease (6 °C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues. CONCLUSIONS: The method and conditions of tumor dissociation influence cell yield and transcriptome state and are both tissue- and cell-type dependent. Interpretation of stress pathway expression differences in cancer single-cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with the identification of such effects in dissociated scRNA-seq experiments.


Assuntos
Genômica/métodos , Neoplasias/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Animais , Temperatura Baixa , Colagenases , Humanos , Camundongos , Peptídeo Hidrolases , Estresse Fisiológico , Transcriptoma
17.
Artigo em Inglês | MEDLINE | ID: mdl-30833417

RESUMO

We report a case of early-onset pancreatic ductal adenocarcinoma in a patient harboring biallelic MUTYH germline mutations, whose tumor featured somatic mutational signatures consistent with defective MUTYH-mediated base excision repair and the associated driver KRAS transversion mutation p.Gly12Cys. Analysis of an additional 730 advanced cancer cases (N = 731) was undertaken to determine whether the mutational signatures were also present in tumors from germline MUTYH heterozygote carriers or if instead the signatures were only seen in those with biallelic loss of function. We identified two patients with breast cancer each carrying a pathogenic germline MUTYH variant with a somatic MUTYH copy loss leading to the germline variant being homozygous in the tumor and demonstrating the same somatic signatures. Our results suggest that monoallelic inactivation of MUTYH is not sufficient for C:G>A:T transversion signatures previously linked to MUTYH deficiency to arise (N = 9), but that biallelic complete loss of MUTYH function can cause such signatures to arise even in tumors not classically seen in MUTYH-associated polyposis (N = 3). Although defective MUTYH is not the only determinant of these signatures, MUTYH germline variants may be present in a subset of patients with tumors demonstrating elevated somatic signatures possibly suggestive of MUTYH deficiency (e.g., COSMIC Signature 18, SigProfiler SBS18/SBS36, SignatureAnalyzer SBS18/SBS36).


Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal Pancreático/genética , DNA Glicosilases/genética , Mutação , Neoplasias Pancreáticas/genética , Idade de Início , DNA Glicosilases/deficiência , Feminino , Mutação em Linhagem Germinativa , Humanos , Perda de Heterozigosidade , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas p21(ras)/genética
18.
Stem Cell Reports ; 10(1): 196-211, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29233553

RESUMO

Human breast cancer cells are known to activate adjacent "normal-like" cells to enhance their own growth, but the cellular and molecular mechanisms involved are poorly understood. We now show by both phenotypic and functional measurements that normal human mammary progenitor cells are significantly under-represented in the mammary epithelium of patients' tumor-adjacent tissue (TAT). Interestingly, fibroblasts isolated from TAT samples showed a reduced ability to support normal EGF-stimulated mammary progenitor cell proliferation in vitro via their increased secretion of transforming growth factor ß. In contrast, TAT fibroblasts promoted the proliferation of human breast cancer cells when these were co-transplanted in immunodeficient mice. The discovery of a common stromal cell-mediated mechanism that has opposing growth-suppressive and promoting effects on normal and malignant human breast cells and also extends well beyond currently examined surgical margins has important implications for disease recurrence and its prevention.


Assuntos
Neoplasias da Mama/metabolismo , Fibroblastos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Neoplasias da Mama/patologia , Feminino , Fibroblastos/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/patologia , Células Estromais/metabolismo , Células Estromais/patologia , Fator de Crescimento Transformador beta/metabolismo
19.
Genome Biol ; 18(1): 140, 2017 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-28750660

RESUMO

Somatic evolution of malignant cells produces tumors composed of multiple clonal populations, distinguished in part by rearrangements and copy number changes affecting chromosomal segments. Whole genome sequencing mixes the signals of sampled populations, diluting the signals of clone-specific aberrations, and complicating estimation of clone-specific genotypes. We introduce ReMixT, a method to unmix tumor and contaminating normal signals and jointly predict mixture proportions, clone-specific segment copy number, and clone specificity of breakpoints. ReMixT is free, open-source software and is available at http://bitbucket.org/dranew/remixt .


Assuntos
Neoplasias da Mama/genética , Cistadenocarcinoma Seroso/genética , Genoma Humano , Modelos Estatísticos , Neoplasias Ovarianas/genética , Software , Algoritmos , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Contagem de Células , Células Clonais , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Variações do Número de Cópias de DNA , Feminino , Genótipo , Xenoenxertos/metabolismo , Xenoenxertos/patologia , Humanos , Internet , Camundongos , Camundongos SCID , Células Neoplásicas Circulantes , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Translocação Genética , Sequenciamento Completo do Genoma
20.
Artigo em Inglês | MEDLINE | ID: mdl-28877932

RESUMO

Whole-genome and transcriptome sequencing were performed to identify potential therapeutic strategies in the absence of viable treatment options for a patient initially diagnosed with vulvar adenocarcinoma. Genomic events were prioritized by comparison against variant distributions in the TCGA pan-cancer data set and complemented with detailed transcriptome sequencing and copy-number analysis. These findings were considered against published scientific literature in order to evaluate the functional effects of potentially relevant genomic events. Analysis of the transcriptome against a background of 27 TCGA cancer types led to reclassification of the tumor as a primary HER2+ mammary-like adenocarcinoma of the vulva. This revised diagnosis was subsequently confirmed by follow-up immunohistochemistry for a mammary-like adenocarcinoma. The patient was treated with chemotherapy and targeted therapies for HER2+ breast cancer. The detailed pathology and genomic findings of this case are presented herein.


Assuntos
Adenocarcinoma/genética , Vulva/patologia , Neoplasias Vulvares/genética , Mama/patologia , Neoplasias da Mama/genética , Diagnóstico Diferencial , Feminino , Perfilação da Expressão Gênica , Genômica , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Transcriptoma , Sequenciamento Completo do Genoma
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