RESUMEN
It is estimated that only 0.02% of disseminated tumour cells are able to seed overt metastases1. While this suggests the presence of environmental constraints to metastatic seeding, the landscape of host factors controlling this process remains largely unclear. Here, combining transposon technology2 and fluorescence niche labelling3, we developed an in vivo CRISPR activation screen to systematically investigate the interactions between hepatocytes and metastatic cells. We identify plexin B2 as a critical host-derived regulator of liver colonization in colorectal and pancreatic cancer and melanoma syngeneic mouse models. We dissect a mechanism through which plexin B2 interacts with class IV semaphorins on tumour cells, leading to KLF4 upregulation and thereby promoting the acquisition of epithelial traits. Our results highlight the essential role of signals from the liver parenchyma for the seeding of disseminated tumour cells before the establishment of a growth-promoting niche. Our findings further suggest that epithelialization is required for the adaptation of CRC metastases to their new tissue environment. Blocking the plexin-B2-semaphorin axis abolishes metastatic colonization of the liver and therefore represents a therapeutic strategy for the prevention of hepatic metastases. Finally, our screening approach, which evaluates host-derived extrinsic signals rather than tumour-intrinsic factors for their ability to promote metastatic seeding, is broadly applicable and lays a framework for the screening of environmental constraints to metastasis in other organs and cancer types.
Asunto(s)
Sistemas CRISPR-Cas , Hepatocitos , Neoplasias Hepáticas , Hígado , Metástasis de la Neoplasia , Proteínas del Tejido Nervioso , Animales , Femenino , Humanos , Masculino , Ratones , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Sistemas CRISPR-Cas/genética , Modelos Animales de Enfermedad , Elementos Transponibles de ADN , Fluorescencia , Hepatocitos/metabolismo , Hepatocitos/citología , Hepatocitos/patología , Factor 4 Similar a Kruppel/metabolismo , Hígado/citología , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/secundario , Melanoma/metabolismo , Melanoma/patología , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/prevención & control , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Semaforinas/antagonistas & inhibidores , Semaforinas/metabolismoRESUMEN
Genome engineering technologies based on CRISPR-Cas systems are fueling efforts to study genotype-phenotype relationships in a high-throughput and multiplexed fashion. While many genome engineering technologies exist and provide a means to efficiently manipulate one or a few genes in a singular context-knockout, inhibition, or activation in a constitutive, conditional, or inducible manner-progress towards engineering complex cellular programs has been hampered by the lack of technologies that can integrate these functions within a unified framework. To address this challenge, our lab created single transcript CRISPR-Cas12a (SiT-Cas12a), which enables conditional, inducible, orthogonal, and massively multiplexed genome engineering of dozens, to potentially hundreds, of genomic targets in eukaryotic cells simultaneously-providing a novel way to interrogate and engineer complex genetic programs. In this chapter, we outline the utility of SiT-Cas12a in human cells and describe experimental procedures for executing massively multiplexed genome engineering experiments-including strategies for designing and assembling customized multiplexed CRISPR guide RNA arrays as well as validating and analyzing CRISPR guide RNA array processing and genome engineering outcomes.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endodesoxirribonucleasas/genética , Edición Génica , Regulación de la Expresión Génica , Transcripción Genética , Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Técnicas de Cultivo de Célula , Endodesoxirribonucleasas/metabolismo , Células HEK293 , Humanos , Reacción en Cadena de la Polimerasa , TransfecciónRESUMEN
Synthesis of ligand-functionalized nanomaterials with control over size, shape, and ligand orientation facilitates the design of targeted nanomedicines for therapeutic purposes. DNA nanotechnology has emerged as a powerful tool to rationally construct two- and three-dimensional nanostructures, enabling site-specific incorporation of protein ligands with control over stoichiometry and orientation. To efficiently target cell surface receptors, exploration of the parameters that modulate cellular accessibility of these nanostructures is essential. In this study, we systematically investigate tunable design parameters of antibody-functionalized DNA nanostructures binding to therapeutically relevant receptors, including the programmed cell death protein 1, the epidermal growth factor receptor, and the human epidermal growth factor receptor 2. We show that, although the native affinity of antibody-functionalized DNA nanostructures remains unaltered, the absolute number of bound surface receptors is lower compared to soluble antibodies due to receptor accessibility by the nanostructure. We explore structural determinants of this phenomenon to improve efficiency, revealing that receptor binding is mainly governed by nanostructure size and DNA handle location. The obtained results provide key insights in the ability of ligand-functionalized DNA nanostructures to bind surface receptors and yields design rules for optimal cellular targeting.