RESUMO
Our knowledge of copy number evolution during the expansion of primary breast tumours is limited1,2. Here, to investigate this process, we developed a single-cell, single-molecule DNA-sequencing method and performed copy number analysis of 16,178 single cells from 8 human triple-negative breast cancers and 4 cell lines. The results show that breast tumours and cell lines comprise a large milieu of subclones (7-22) that are organized into a few (3-5) major superclones. Evolutionary analysis suggests that after clonal TP53 mutations, multiple loss-of-heterozygosity events and genome doubling, there was a period of transient genomic instability followed by ongoing copy number evolution during the primary tumour expansion. By subcloning single daughter cells in culture, we show that tumour cells rediversify their genomes and do not retain isogenic properties. These data show that triple-negative breast cancers continue to evolve chromosome aberrations and maintain a reservoir of subclonal diversity during primary tumour growth.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células , Células Clonais/metabolismo , Células Clonais/patologia , Evolução Molecular , Sequência de Bases , Linhagem Celular Tumoral , Linhagem da Célula , Aberrações Cromossômicas , Variações do Número de Cópias de DNA/genética , Análise Mutacional de DNA , Instabilidade Genômica/genética , Humanos , Perda de Heterozigosidade/genética , Modelos Genéticos , Taxa de Mutação , Imagem Individual de Molécula , Análise de Célula Única , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Immunotherapy has transformed cancer treatment. However, current immunotherapy modalities face various limitations. In the present study, we developed multiplexed activation of endogenous genes as an immunotherapy (MAEGI), a new form of immunotherapy that elicits antitumor immunity through multiplexed activation of endogenous genes in tumors. We leveraged CRISPR activation (CRISPRa) to directly augment the in situ expression of endogenous genes, and thereby the presentation of tumor antigens, leading to dramatic antitumor immune responses. Deploying this as a cell-based vaccination strategy showed efficacy in both prophylactic and therapeutic settings. Intratumoral adeno-associated virus delivery of CRISPRa libraries elicited strong antitumor immunity across multiple cancer types. Precision targeting of mutated gene sets eradicated a large fraction of established tumors at both local and distant sites. This treatment modality led to alterations in the tumor microenvironment, marked by enhanced T cell infiltration and antitumor immune signatures. Multiplexed endogenous gene activation is a versatile and highly scalable strategy to elicit potent immune responses against cancer, distinct from all existing cancer therapies.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Terapia Genética/métodos , Imunoterapia/métodos , Neoplasias/tratamento farmacológico , Animais , Apresentação de Antígeno/genética , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Terapia Combinada/métodos , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Células HEK293 , Humanos , Injeções Intralesionais , Linfócitos do Interstício Tumoral/imunologia , Masculino , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
The genetic makeup of cancer cells directs oncogenesis and influences the tumor microenvironment. In this study, we massively profiled genes that functionally drive tumorigenesis using genome-scale in vivo CRISPR screens in hosts with different levels of immunocompetence. As a convergent hit from these screens, Prkar1a mutant cells are able to robustly outgrow as tumors in fully immunocompetent hosts. Functional interrogation showed that Prkar1a loss greatly altered the transcriptome and proteome involved in inflammatory and immune responses as well as extracellular protein production. Single-cell transcriptomic profiling and flow cytometry analysis mapped the tumor microenvironment of Prkar1a mutant tumors and revealed the transcriptomic alterations in host myeloid cells. Taken together, our data suggest that tumor-intrinsic mutations in Prkar1a lead to drastic alterations in the genetic program of cancer cells, thereby remodeling the tumor microenvironment.
