RESUMEN
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.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular , Células Clonales/metabolismo , Células Clonales/patología , Evolución Molecular , Secuencia de Bases , Línea Celular Tumoral , Linaje de la Célula , Aberraciones Cromosómicas , Variaciones en el Número de Copia de ADN/genética , Análisis Mutacional de ADN , Inestabilidad Genómica/genética , Humanos , Pérdida de Heterocigocidad/genética , Modelos Genéticos , Tasa de Mutación , Imagen Individual de Molécula , Análisis de la Célula Individual , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
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.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Regulación Neoplásica de la Expresión Génica/inmunología , Terapia Genética/métodos , Inmunoterapia/métodos , Neoplasias/tratamiento farmacológico , Animales , Presentación de Antígeno/genética , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Técnicas de Cocultivo , Terapia Combinada/métodos , Dependovirus/genética , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Células HEK293 , Humanos , Inyecciones Intralesiones , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Ratones , Neoplasias/genética , Neoplasias/inmunología , Linfocitos T Citotóxicos/inmunología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
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.
