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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.
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
3.
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
4.
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
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