RESUMO
BACKGROUND: MicroRNAs (miRNAs) are key regulators of stem cell functions, including self-renewal and differentiation. In this study, we aimed to identify miRNAs that are upregulated during terminal differentiation in the human colon epithelium, and elucidate their role in the mechanistic control of stem cell properties. METHODS: "Bottom-of-the-crypt" (EPCAM+/CD44+/CD66alow) and "top-of-the-crypt" (EPCAM+/CD44neg/CD66ahigh) epithelial cells from 8 primary colon specimens (6 human, 2 murine) were purified by flow cytometry and analyzed for differential expression of 335 miRNAs. The miRNAs displaying the highest upregulation in "top-of-the-crypt" (terminally differentiated) epithelial cells were tested for positive correlation and association with survival outcomes in a colon cancer RNA-seq database (n = 439 patients). The two miRNAs with the strongest "top-of-the-crypt" expression profile were evaluated for capacity to downregulate self-renewal effectors and inhibit in vitro proliferation of colon cancer cells, in vitro organoid formation by normal colon epithelial cells and in vivo tumorigenicity by patient-derived xenografts (PDX). RESULTS: Six miRNAs (miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-345) were upregulated in "top-of-the-crypt" cells and positively correlated in expression among colon carcinomas. Overexpression of the three miRNAs with the highest inter-correlation coefficients (miR-200a, miR-200b, miR-200c) associated with improved survival. The top two over-expressed miRNAs (miR-200c, miR-203) cooperated synergistically in suppressing expression of BMI1, a key regulator of self-renewal in stem cell populations, and in inhibiting proliferation, organoid-formation and tumorigenicity of colon epithelial cells. CONCLUSION: In the colon epithelium, terminal differentiation associates with the coordinated upregulation of miR-200c and miR-203, which cooperate to suppress BMI1 and disable the expansion capacity of epithelial cells.
Assuntos
Neoplasias do Colo , MicroRNAs , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , MicroRNAs/genética , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas , Regulação para CimaRESUMO
Organoid culture is a three-dimensional culture method that enables ex vivo analysis of stem cell behavior and differentiation. This method is also applicable to the studies on stem cell characters of human cancer stem cells. The components of organoid culture include Matrigel® and a culture medium containing growth factor cocktails that mimic the microenvironments of organ stem cell niches. Here, we describe the basic methods for the organoid culture of dissociated or FACS-sorted human cancer stem cells. Then, we introduce a method to dissociate the organoids for serial passage and propagation.
Assuntos
Técnicas de Cultura de Células/métodos , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Organoides/patologia , Diferenciação Celular , Humanos , Nicho de Células-Tronco , Células Tumorais CultivadasRESUMO
It has been postulated that there is a link between inflammation and cancer. Here we describe a role for cell-intrinsic toll-like receptor-2 (TLR2; which is involved in inflammatory response) signalling in normal intestinal and mammary epithelial cells and oncogenesis. The downstream effectors of TLR2 are expressed by normal intestinal and mammary epithelia, including the stem/progenitor cells. Deletion of MYD88 or TLR2 in the intestinal epithelium markedly reduces DSS-induced colitis regeneration and spontaneous tumour development in mice. Limiting dilution transplantations of breast epithelial cells devoid of TLR2 or MYD88 revealed a significant decrease in mammary repopulating unit frequency compared with the control. Inhibition of TLR2, its co-receptor CD14, or its downstream targets MYD88 and IRAK1 inhibits growth of human breast cancers in vitro and in vivo. These results suggest that inhibitors of the TLR2 pathway merit investigation as possible therapeutic and chemoprevention agents.
Assuntos
Neoplasias da Mama/patologia , Mama/patologia , Carcinogênese/metabolismo , Neoplasias do Colo/patologia , Mucosa Intestinal/patologia , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 2 Toll-Like/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Mama/metabolismo , Neoplasias da Mama/genética , Carcinogênese/genética , Moléculas de Adesão Celular/metabolismo , Colite/induzido quimicamente , Colite/patologia , Neoplasias do Colo/genética , Sulfato de Dextrana , Molécula de Adesão da Célula Epitelial , Epitélio/patologia , Feminino , Células HEK293 , Humanos , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Quinases Associadas a Receptores de Interleucina-1/genética , Mucosa Intestinal/metabolismo , Antígenos Comuns de Leucócito/genética , Receptores de Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Fator 88 de Diferenciação Mieloide/genética , Transplante de Neoplasias , Interferência de RNA , RNA Interferente Pequeno , Receptores Acoplados a Proteínas G/metabolismo , Regeneração/genética , Transdução de Sinais , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/genética , Células Tumorais CultivadasRESUMO
In multicellular organisms and complex ecosystems, cells migrate in a social context. Whereas this is essential for the basic processes of life, the influence of neighboring cells on the individual remains poorly understood. Previous work on isolated cells has observed a stereotypical migratory behavior characterized by short-time directional persistence with long-time random movement. We discovered a much richer dynamic in the social context, with significant variations in directionality, displacement, and speed, which are all modulated by local cell density. We developed a mathematical model based on the experimentally identified "cellular traffic rules" and basic physics that revealed that these emergent behaviors are caused by the interplay of single-cell properties and intercellular interactions, the latter being dominated by a pseudopod formation bias mediated by secreted chemicals and pseudopod collapse following collisions. The model demonstrates how aspects of complex biology can be explained by simple rules of physics and constitutes a rapid test bed for future studies of collective migration of individual cells.
Assuntos
Comunicação Celular/fisiologia , Movimento Celular/fisiologia , Modelos Biológicos , Movimento/fisiologia , Fenômenos Biofísicos , Contagem de Células , MicrofluídicaRESUMO
Cancer is often viewed as a caricature of normal developmental processes, but the extent to which its cellular heterogeneity truly recapitulates multilineage differentiation processes of normal tissues remains unknown. Here we implement single-cell PCR gene-expression analysis to dissect the cellular composition of primary human normal colon and colon cancer epithelia. We show that human colon cancer tissues contain distinct cell populations whose transcriptional identities mirror those of the different cellular lineages of normal colon. By creating monoclonal tumor xenografts from injection of a single (n = 1) cell, we demonstrate that the transcriptional diversity of cancer tissues is largely explained by in vivo multilineage differentiation and not only by clonal genetic heterogeneity. Finally, we show that the different gene-expression programs linked to multilineage differentiation are strongly associated with patient survival. We develop two-gene classifier systems (KRT20 versus CA1, MS4A12, CD177, SLC26A3) that predict clinical outcomes with hazard ratios superior to those of pathological grade and comparable to those of microarray-derived multigene expression signatures.