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1.
STAR Protoc ; 2(4): 100780, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34585151

RESUMO

Patient-derived organoids (PDOs) recapitulate the cellular heterogeneity of the original colorectal tumor tissue. Here, we describe a protocol to generate genetically modified PDOs to investigate cancer stem cells. This protocol uses the CRISPR-Cas9 system to knock-in the IRES-EGFP-P2A-iCaspase9 cassette into the 3' UTR of the potential cancer stem cell marker gene, which allows us to investigate their potential for self-replication and pluripotency. We describe the procedure for generating mutant PDOs and their application for stem cell research. For complete details on the generation and use of this protocol, please refer to Okamoto et al. Okamoto et al. (2021).


Assuntos
Sistemas CRISPR-Cas/genética , Neoplasias Colorretais , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Organoides/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Células-Tronco Neoplásicas/citologia , Células Tumorais Cultivadas
2.
Stem Cell Reports ; 16(4): 954-967, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33711267

RESUMO

Metastasis is the major cause of cancer-related death, but whether metastatic lesions exhibit the same cellular composition as primary tumors has yet to be elucidated. To investigate the cellular heterogeneity of metastatic colorectal cancer (CRC), we established 72 patient-derived organoids (PDOs) from 21 patients. Combined bulk transcriptomic and single-cell RNA-sequencing analysis revealed decreased gene expression of markers for differentiated cells in PDOs derived from metastatic lesions. Paradoxically, expression of potential intestinal stem cell markers was also decreased. We identified OLFM4 as the gene most strongly correlating with a stem-like cell cluster, and found OLFM4+ cells to be capable of initiating organoid culture growth and differentiation capacity in primary PDOs. These cells were required for the efficient growth of primary PDOs but dispensable for metastatic PDOs. These observations demonstrate that metastatic lesions have a cellular composition distinct from that of primary tumors; patient-matched PDOs are a useful resource for analyzing metastatic CRC.


Assuntos
Neoplasias Colorretais/patologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Organoides/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/cirurgia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Neoplásica , Organoides/patologia
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