Background: The definition of cancer stem cells (CSCs) as “tumor cells that contain self-renewal and multilineage differentiation capacity” still lacks conclusive experimental evidence. This is partly because the gold standard assay to identify CSCs, serial transplantation in animal models, is an operation that can undervalue total CSC numbers. Recently, the combination of mouse models that spontaneously develop tumors with genetic tracing provided exciting support for the CSC theory. Here, we took advantage of the use of cell-surface biomarkers, to purify, to some extent, murine cell populations with CSC phenotype by cell sorting on fresh spontaneous colorectal cancer (CRC). The aim of our study was to define a panel of miRNAs pathways with a potential function in the orchestration of selfrenewal and cell fate during cancer development using tissue of colorectal cancer from both mouse and human origin. Methods: Different populations of murine colon cells derived from AOM/DSS-induced carcinoma were isolated with cell sorting performed on a BD FACSAria system, by the expression of cell surface biomarkers (CD44, EphB2), then stem/progenitor cells-like property was analysed by the expression of stemness/differentiation genes (gene expression and IHC analysis). Specific regions of AOM/DSS treated colon mucosa containing dysplastic ACFs, microadenoma, adenoma, carcinoma compared to normal mucosa were isolated with Laser Capture Microdissection for RNA extraction. miRNAs expression profiling were performed using TaqMan Low Density Arrays (LifeTechnologies) both on cells and tissue samples. Microarray data were evaluated by state of the art statistics and bioinformatics tools. Single Real-Time PCR for selected miRNAs were performed on a training set of 60 FFPE human samples. Results: Global miRNAs expression profiles showed a total of ∼100 miRNAs with a significantly (p<0,05) up/down regulation in a gradual or punctual way in 4 different stages of murine CRC. Numerous miRNAs were already demonstrated to be deregulated in hCRC, confirming the high reliability of the murine model to mimic the human pathology. A set of ∼100 miRNAs distinguished the 4 steps of CRC progression in a unsupervised hierarchical clustering analysis (p<0,05). The analysis of miRNAs expression pattern in the sorted stem-like cell populations in comparison to miRNAs altered in the murine CRC initiation/progression steps revealed a significant cancer stemness signature, composed by miRNAs related to pluripotency and differentiation. Some of these changes, including miR-200b, mir-429, mir-365, mir-215, mir-26b, mir-135, were studied as well in the human samples, showing a similar pattern of that observed in the murine model. Conclusions: These data provide a comprehensive miRNAs signature implicated in the regulation of tumorigenesis, stemness, and cell fate determination that could be exploited for diagnosis and therapeutic design.
Phase specific microRNA deregulation of oncogenesis and stemness in a mouse model of sporadic CRC
Fazio V. M.
2014-01-01
Abstract
Background: The definition of cancer stem cells (CSCs) as “tumor cells that contain self-renewal and multilineage differentiation capacity” still lacks conclusive experimental evidence. This is partly because the gold standard assay to identify CSCs, serial transplantation in animal models, is an operation that can undervalue total CSC numbers. Recently, the combination of mouse models that spontaneously develop tumors with genetic tracing provided exciting support for the CSC theory. Here, we took advantage of the use of cell-surface biomarkers, to purify, to some extent, murine cell populations with CSC phenotype by cell sorting on fresh spontaneous colorectal cancer (CRC). The aim of our study was to define a panel of miRNAs pathways with a potential function in the orchestration of selfrenewal and cell fate during cancer development using tissue of colorectal cancer from both mouse and human origin. Methods: Different populations of murine colon cells derived from AOM/DSS-induced carcinoma were isolated with cell sorting performed on a BD FACSAria system, by the expression of cell surface biomarkers (CD44, EphB2), then stem/progenitor cells-like property was analysed by the expression of stemness/differentiation genes (gene expression and IHC analysis). Specific regions of AOM/DSS treated colon mucosa containing dysplastic ACFs, microadenoma, adenoma, carcinoma compared to normal mucosa were isolated with Laser Capture Microdissection for RNA extraction. miRNAs expression profiling were performed using TaqMan Low Density Arrays (LifeTechnologies) both on cells and tissue samples. Microarray data were evaluated by state of the art statistics and bioinformatics tools. Single Real-Time PCR for selected miRNAs were performed on a training set of 60 FFPE human samples. Results: Global miRNAs expression profiles showed a total of ∼100 miRNAs with a significantly (p<0,05) up/down regulation in a gradual or punctual way in 4 different stages of murine CRC. Numerous miRNAs were already demonstrated to be deregulated in hCRC, confirming the high reliability of the murine model to mimic the human pathology. A set of ∼100 miRNAs distinguished the 4 steps of CRC progression in a unsupervised hierarchical clustering analysis (p<0,05). The analysis of miRNAs expression pattern in the sorted stem-like cell populations in comparison to miRNAs altered in the murine CRC initiation/progression steps revealed a significant cancer stemness signature, composed by miRNAs related to pluripotency and differentiation. Some of these changes, including miR-200b, mir-429, mir-365, mir-215, mir-26b, mir-135, were studied as well in the human samples, showing a similar pattern of that observed in the murine model. Conclusions: These data provide a comprehensive miRNAs signature implicated in the regulation of tumorigenesis, stemness, and cell fate determination that could be exploited for diagnosis and therapeutic design.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
