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Isolation and genomic characterization of cancer stem cells in head and neck cancer

ABSTRACT: This study investigated the use of three different established cell sorting strategies to isolate and characterize stem cells from head and neck cancer cell lines. Five low passage cell lines were subjected to cell sorting based on Hoechst side population, Aldefluor and CD44 expression. Isolated cell populations were studied for gene expression, radiosensitivity and chemosensitivity to cis-platin and paclitaxel Each sorting method identified a different set of genes associated with different Gene Ontology categories with mitosis being the only common category. CD44 associated gene changes were almost exclusively associated with cell cycle and in particular mitosis. There were no significant differences in radiosensitivity or cis-platin sensitivity of stem or non-stem cells but CD44 isolated stem cells were more resistant to paclitaxel

ORGANISM(S): Homo sapiens

PROVIDER: GSE55487 | GEO | 2014/03/02

SECONDARY ACCESSION(S): PRJNA239762

REPOSITORIES: GEO

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Project description:Abstract: The intestinal epithelium is replaced weekly by non-quiescent stem cells with kinetics that rely on a rapid loss of stemness and choice for secretory or absorptive lineage differentiation. To determine how the cellular transcriptome and proteome changes during these transitions, we developed a new cell sorting method to purify stem cells, secretory and absorptive progenitor cells, and mature, differentiated cells. Transcriptome analyses revealed that as stem cells transition to the progenitor stage, alternative mRNA splicing and polyadenylation dominate changes in the transcriptome. In contrast, as progenitors differentiate into mature cell types, alterations in gene expression and mRNA levels drive the changes. RNA processing targets mRNAs encoding regulators of cell cycle, RNA regulators, cell adhesion, SUMOylation, and Wnt and Notch signaling. Additionally, carrier-assisted mass spectrometry of sorted cell populations detected >2,800 proteins and revealed RNA:protein patterns of abundance and correlation. Paired together, these data highlight new potentials for autocrine and feedback regulation and provide new insights into cell state transitions in the crypt. Sorting Protocol: To create a high-resolution profile of colon crypt stem cells and their daughter cells we developed a new flow sorting protocol using freshly dissected, wild-type C57Bl/6N mouse colons and antibodies to validated intestinal cell surface markers including Cd44. Upon discovery that Cd44 is highly sensitive to TrypLE, and other commonly used proteases, we developed a dissociation protocol that uses only EDTA and mechanical force. This change enabled a 10-fold greater range of Cd44 antigen surface expression and therefore higher resolution for cell sorting. Using additional commonly used cell surface markers, six cryptal populations were purified. A previously validated intestinal stem cell signature of Cd44 high, Cd24 low, and cKit negative was used to identify and isolate an abundant fraction of stem cells, a cell population that directly overlapped with Lgr5-EGFP+ cells from Lgr5-EGFP-IRES-creERT2 mice, confirming their stem cell identity. In addition to the stem cell population, five additional Epcam positive populations were collected, and the biological replicate samples of the six populations were processed for bulk RNA-seq. The clearly identified populations are stem cells, two distinct populations of progenitor cells (absorptive and secretory), and three mature, differentiated populations (enterocytes, Tuft cells and Enteroendocrine (EEC) cells). Thus, the new protocol for crypt isolation and the greater range of Cd44 surface expression it preserves, enables a significant improvement in the resolution and sorting of stem cells away from their daughter cells and differentiated progeny. Specifically, it is now possible to distinguish stem cells from Absorptive Progenitor cells (AbsPro; Cd44Med) and from mature Enterocytes (Ent; Cd44Low/-) because of the higher resolution of Cd44 surface expression. Secretory progenitors are identified as SecPDG as this population contains a majority of Secretory Progenitors and Deep Crypt Secretory cells, with a possible minor contribution of Goblet cells, a cell type that is largely missing in this procedure. Finally, preserved Cd44 expression (and cKit expression) enabled resolution of two rare Epcam+/Cd24high populations identified as Tuft cells and Enteroendocrine cells (EEC), mature cell types from the secretory lineage (EEC are predominantly Enterochromaffin; Tuft, comprise both Tuft-1 and Tuft-2 subtypes).

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Isolation and genomic characterization of cancer stem cells in head and neck cancer

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